CN103272225B - The oxalate decarboxylase and application method of crystallization - Google Patents

Abstract

Oxalate decarboxylase crystal is disclosed, including stabilized crystal, such as crosslinked oxalate decarboxylase crystal.Also disclose that the method that the illness related with raised concentration of oxalate is treated using oxalate decarboxylase crystal.In addition, disclose the method for preparing albumin crystal.

Description

The oxalate decarboxylase and application method of crystallization

The application is Chinese Patent Application No. 200780035792.4（PCT/US2007/075091）, at 2007 8 applying date The moon 2, the divisional application of entitled " oxalate decarboxylase and application method of crystallization "

The cross reference of related application

The U.S.Application Serial Number 60/834,933 submitted for 2nd this application claims August in 2006 and on October 26th, 2006 The priority of the U.S.Application Serial Number 60/854,540 of submission, their content is by reference to being integrally incorporated herein.

Background technology

Oxalic acid is formula HO₂C-CO₂The dicarboxylic acids of H.Oxalic acid is present in mainly as oxalates in organism, the oxalates It is the salt form of oxalic acid.Oxalates is seen in food, for example, spinach, rheum officinale, strawberry, european cranberry, nut, cocoa, chocolate, Peanut butter, sorghum, and tealeaves.Oxalates is also end product of metabolism in the mankind and other mammals.It is by renal excretion into urine In.When being combined with calcium, oxalic acid generates insoluble product calcium oxalate, it is the most common compound found in kidney stone.

Because mammal will not synthesis and degradation oxalates enzyme, individual in levels of oxalate generally by draining and drinking The low absorption of oxalates is eaten to remain normal.The oxalates of high concentration is related with a variety of pathology, such as primary hyperoxaluria, Intestines originality hyperoxaluria and idiopathic hyperoxaluria.Leumann et al., Nephrol.Dial.Transplant.14: 2556-2558 (1999) and Earnest, Adv.Internal Medicine24:407-427(1979).Increased oxalates Reason can be the oxalates excessive from food intake, and the exception of oxalates and oxalates generation is excessively absorbed from enteron aisle.Grass Absorption of the hydrochlorate in colon and small intestine is excessively related with enteropathy, including the suction as caused by bile acid and fat absorption bad disease It receives excessive；Ileal resection;With for example, the steatorrhea as caused by chylous diarrhea, exocrine pancreas are insufficient, enteropathy and hepatopathy.

Hyperoxaluria or the excretion of increased urinary oxalate are related to calcium oxalate in nephridial tissue (nephrocalcinosis) or urine with many Road is (for example, kidney stone（kidney stones）, lithangiuria, and kidney stone（nephrolithiasis）) in deposition Health problem is related.Calcium oxalate can also be deposited on such as eyes, blood vessel, joint, bone, muscle, heart and other vitals, They are damaged.See, e.g., Leumann et al., J.Am.Soc.Nephrol.12:1986 1993 (2001) and Monico et al., Kidney International62:392400(2002).The influence of increased levels of oxalate can occur In Various Tissues.For example, the deposition in thin vessels can cause to be difficult to the skin ulcer for the pain fully recovered, it is heavy in marrow Product can cause anaemia, and the deposition in bone tissue can cause to fracture or influence the growth of children, the calcium oxalate deposits in heart Heart rate exception or cardiac function can be caused poor.

The method of the existing raised levels of oxalate for the treatment of is not always effective, and many primary hyperoxaluria patients can It can need intensive dialysis and organ transplant.The existing therapy of different hyperoxalurias include high dose vitamin B6, orthophosphates, Magnesium, iron, aluminium, potassium citrate, Cholestyramine and glycosaminoglycan treatment and adjusting diet and fluid intake, dialysis and surgery hand Art（Such as kidney and liver transfer operation）Scheme.These therapies are (for example, low oxalates or low fat diet, vitamin B6, enough calcium With increased fluid) it is only partially effective, they may have undesirable adverse side effect, such as orthophosphates, magnesium Or Cholestyramine supplement gastrointestinal effects and dialysis and operation risk.Therefore, it is necessary to safely from body removal oxalates Method.In addition, degrading oxalates to reduce the method for the levels of oxalate in biological sample, surpass such as independent blocking oxalates Absorption or accelerate oxalates remove therapy.

The content of the invention

The present invention relates to oxalate decarboxylase (" OXDC ") crystal and its cross-linked form (" CLEC ") and they be used to treat it is all Such as the purposes of the oxalates associated conditions of hyperoxaluria.In one embodiment, lenticular oxalate decarboxylase can be administered to Mammal, for example, it is oral or be applied directly to stomach, to reduce levels of oxalate and/or reduce calcium oxalate in mammals Damage caused by deposition.In addition, disclose the method from cell extract production albumin crystal.It also discloses that comprising oxalic acid decarboxylation The composition of enzyme (" OXDC ") crystal and its cross-linked form (" CLEC "), for example, pharmaceutical composition.

In one aspect, the present invention provides crosslinked oxalate decarboxylase crystal.Crosslinking agent can be it is multi-functional, In some embodiments, which is bifunctional reagent, such as glutaraldehyde.In certain embodiments, oxalate decarboxylase crystal The concentration of glutaraldehyde cross-linking with not substantially changing concentration of enzymatic activity, for example, at least about 0.02% (w/v).In embodiments, The cross-linking level of oxalate decarboxylase crystal is equal to the level for being handled and being generated with 0.02% (w/v) glutaraldehyde.Cross-linking level can pass through Known in the art or method disclosed herein measures, for example, the level of albumen leaching is measured, such as such as embodiment 10-11 Disclosed.

The present invention also provides oxalate decarboxylase crystal, for example, with higher than soluble oxalate decarboxylase for example, at least about 100%th, the oxalate decarboxylase crystal of 200%, 300%, 400% or 500% activity.

The present invention also provides stabilized, for example crosslinked oxalate decarboxylase crystal, wherein the stabilized crystal The activity and/or stability retained in acid condition is than soluble oxalate decarboxylase in similar acid condition (for example, about 2 to 3 Acid pH) under activity and/or at least 2,3 times of stability height.In embodiments, the stabilized oxalate decarboxylase is brilliant Body is higher by least 200% than the activity and/or stability of soluble oxalate decarboxylase in acid condition, 300%, 400%.

The present invention also provides stabilized, for example crosslinked oxalate decarboxylase crystal, wherein the stabilized crystal The activity and/or stability retained in the presence of protease is than activity that soluble oxalate decarboxylase retains under similar conditions And/or at least 2,3 times of stability height.In embodiments, the stabilized oxalate decarboxylase crystal takes off than soluble oxalic acid Activity and/or stability height at least 200%, 300%, 400% of the carboxylic acid in the presence of protease.The protease can be selected from Following one or more：For example, pepsin, chymotrypsin or pancreatin.In embodiments, by the stabilisation Crystal or soluble oxalate decarboxylase exposed to acid condition and/or the predetermined time span for example, at least 1 of protease, 2,3, After 4 or 5 hours, the activity (for example, as described in embodiment hereof) of described stabilized or soluble oxalate decarboxylase is measured.

In terms of one related, the present invention characterizes a kind of crosslinked oxalate decarboxylase crystal, it is in condition of different pH Under (for example, about pH2.5 or 3 to 7.5 or 8.5) and/or in the presence of protease be substantially it is active and be it is stable, For example, protease can be selected from following one or more：For example, pepsin, chymotrypsin or pancreatin.Such as this paper institutes State, in embodiments, the activity that the crosslinked crystal retains than soluble oxalate decarboxylase in acid condition (for example, About 2 to 3 acid pH) and retain in the presence of protease active at least 2,3 times high.As described herein, in other implementations In scheme, the stabilized oxalate decarboxylase crystal is than soluble oxalate decarboxylase in acid condition (for example, about 2 to 3 Acid pH) and stability height at least 200%, 300%, 400% in the presence of protease.

Composition comprising crystal as described herein and/or crosslinked oxalate decarboxylase crystal, for example, pharmaceutical composition, Also within the scope of the present invention.

In some embodiments, the crystal includes having and the sequence of the oxalate decarboxylase found in natural origin The oxalate decarboxylase of identical or substantially the same sequence, the especially natural origin such as plant, bacterium and fungi, withered grass gemma Bacillus, needle mushroom or flammulina velutipes, aspergillus niger, pseudomonas（synechoystis sp.）, cyanobacteria category, streptococcus mutans, Trametes trogii（Trametes hirsute）, sclerotinite, whiterot fungi（T.versicolor）, brown rot fungus（Postia placenta）, Myrothecium verrucaria, Agaricus bisporus, methylotrophic bacteria（Methylobacterium extorquens）,Pseudomonas Oxalaticus, Ralstonia eutropha, Cupriavidus oxalaticus, Wautersia sp., Oxalicibacterium flavum,Ammoniiphilus oxalaticus,Vibrio oxalaticus,A.oxalativorans,Variovorax Paradoxus, xanthobacter autotrophicus, Eurotium, Penicillium, and Mucor.In other embodiments, recombinant production Oxalate decarboxylase.

In one aspect, the present invention provides the method for the concentration of oxalate for reducing subject, wherein application is disclosed herein Composition, for example, pharmaceutical composition, it includes oxalate decarboxylase crystal, for example, crosslinked oxalate decarboxylase crystal.One In a embodiment, the crosslinking agents such as described oxalate decarboxylase crystal glutaraldehyde stabilize.The application of composition can make oxalic acid Salinity reduces at least 10%, at least 20%, at least 30% or at least 40% or more.In some embodiments, it is oral or pass through Device outside applying said compositions.In one embodiment, the device outside is conduit, for example, scribbling oxalic acid decarboxylation The conduit of enzyme crystal.In other embodiments, the composition is applied as suspension, dry powder, capsule or tablet.At one In embodiment, the method for the concentration of oxalate for reducing mammal includes measuring the biological sample of mammal（Such as Urine, blood, blood plasma or blood serum sample）In concentration of oxalate the step of.

On the other hand, it is dense with raised oxalates in mammal the present invention provides treating, preventing and/or slowing down The method of the progress of related illness is spent, wherein applying oxalate decarboxylase crystal and/or stabilized, example to the mammal Such as crosslinked oxalate decarboxylase crystal.In one embodiment, the illness related with raised concentration of oxalate is kidney Disease, arthropathy, eye disease, hepatopathy, gastrointestinal disease or Pancreas Disease.In certain embodiments, the illness is the high oxalic acid of primary Urine, intestines originality hyperoxaluria, idiopathic hyperoxaluria, ethylene glycol poisoning, cystic fibrosis, inflammatory bowel disease, lithangiuria, Kidney stone, chronic kidney disease, haemodialysis and gastrointestinal bypass.

On the other hand, the present invention provides a kind of composition, for example, pharmaceutical composition, it includes oxalate decarboxylases Crystal, for example, crosslinked oxalate decarboxylase crystal (for example, crystal disclosed herein and/or crosslinked crystal).

On the other hand, the present invention provides the method for the treatment of mammal, wherein using a effective amount of pharmaceutical composition Object, the latter include oxalate decarboxylase crystal, for example, crosslinked oxalate decarboxylase crystal (for example, crystal disclosed herein and/or Crosslinked crystal).

On the other hand, the present invention provides production albumin crystal such as enzyme crystals (for example, oxalate decarboxylase crystal) Method, including:There is provided the product of the cell extract containing the albumen or particle/precipitation/solution and from the product Crystallize the albumen.In embodiments, the described method includes following one or more steps:The original of the albumen is expressed in culture Core host cell cultures;Obtain the product of the particle containing target protein or extract；Dissolved particles product；Make albumin crystal It is formed and/or crystalchecked is further made by crosslinking.In general, recombinantly express the albumen.In embodiments, particle Product includes inclusion body.

In embodiments, the dissolving step includes, and following one or more are added in into particle product：Comprising mild The solution of denaturant concentration (for example, the urea or guanidine hydrochloride of concentration in for example, about 1M to about 3M);Include high salt concentration（For example, institute State one or more of the salt in sodium chloride, potassium chloride, calcium chloride）Or for example, about 0.3 to about 0.8M concentration it is other The solution of salt;In alkaline conditions（For example, the pH in about 9 to about 12）Include the solution of mild denaturant concentration；Or include height Denaturant concentration（For example, about 4M is to about 8M ureas or guanidine hydrochloride）Solution.

In embodiments, the purification step includes, and fragment is removed from particle, for example, by separating dissolvingization Grain product（For example, pass through one or more rotation or centrifugation step）And/or it collects supernatant.The purification step can be optional Ground further includes, and makes the particle product of dissolving through ion-exchange chromatography and/or filters the product of dissolvingization.

In embodiments, the crystallisation step includes, the albumen of concentrating and purifying, so as to form the albumen of crystallization. In embodiment, the albumen of crystallization is obtained from the supernatant collected after separating step, for example, being rotated in one or more Or after centrifugation step.In addition crystallisation step can include, the albumen of crystallization is made to contact crosslinking agent, for example, friendship disclosed herein Join agent (for example, glutaraldehyde).The concentration of the crosslinking agent used can 0.01% to 20%w/v scope；Usual 0.02% to 10% w/v；More generally 0.02%, 0.5% or 1%w/v.

In embodiments, the yield of albumen is the spy found in the cell product for obtaining particle product in particle product Determine at least about 50%, 60%, 70%, the 80% of albumen.In other embodiments, the yield of the albumen of dissolving is in particle product At least about 90%, the 95% of middle discovery or higher.In other embodiments, the yield of the albumen of crystallization is in particle product It was found that at least about 50%, 60%, 70%, 80%.

The present invention also provides the albumin crystal produced by method disclosed herein, for example, enzyme crystal is (for example, oxalic acid Decarboxylase crystals).

In the the accompanying drawings and the following description, the details of one or more embodiments of the present invention is illustrated.

Description of the drawings

Fig. 1 is the soluble oxalate decarboxylase (" soluble ") of display, oxalate decarboxylase crystal (" crystal ") and crosslinked grass The figure of the pH living features of decarboxylase crystal (" CLEC ").

Fig. 2 is bar chart of the description using the urinary oxalate level in Sprague Dawley rats after OXDC-CLEC.Often A item represents average value ± SE (standard error).Asterisk instruction is examined using double tail Student t and calculated at the specified time point Significant difference p between control group and 3 treatment groups<0.05.

Fig. 3 is the drop for the urinary oxalate level in (KO) mouse that OXDC-CLEC knocks out the AGT1 that ethylene glycol is attacked that describes The bar chart of low influence.Each item represents average value ± SE.Asterisk instruction examines what is calculated referring to using double tail Student t The significant difference p to fix time between a control group and 3 treatment groups<0.05.

Fig. 4 is the item for the influence for describing the creatinine clearance in the AGT1 KO mouse that OXDC-CLEC attacks ethylene glycol Line chart.Each item represents average value ± SE.Asterisk instruction examines the control group calculated and 80mg treatments using double tail Student t Significant difference p between group<0.05.

Fig. 5 A-5C be show treated with OXDC-CLEC after the substantial calcium oxalate deposits object of kidney prevention image.Fig. 5 A Come the section of the kidney essence of the mouse of the EG attacks for 80mg OXDC-CLEC processing of using by oneself.Fig. 5 B and 5C are from control group The section of kidney essence.The section of Fig. 5 B confirms medium nephrocalcinosis, and the section of Fig. 5 C confirms serious nephrocalcinosis.It is deep Color spot is calcium oxalate deposits object (example pointed out with white arrow), and light spot is that grey (is used in the region with interstitial fibrosis The example that arrow is pointed out).

When Fig. 6 is comparison with the survival of the mouse of the OXDC-CLEC or vehicle-control of 3 kinds of various doses the EG attacks handled Between Kaplan-Meier survival figure.

Fig. 7 be describe to be spaced at the appointed time, soluble oxalate decarboxylase (" Sol "), oxalate decarboxylase in low pH The figure of the stability of crystal (" XTAL ") and crosslinked oxalate decarboxylase crystal (" CLEC ").

Fig. 8 is to describe to be spaced at the appointed time, in pH3.0, the soluble oxalic acid decarboxylation in the presence of pepsin The figure of the stability of enzyme (" Sol "), oxalate decarboxylase crystal (" XTAL ") and crosslinked oxalate decarboxylase crystal (" CLEC ").

Fig. 9 be describe to be spaced at the appointed time, in pH7.5, in the presence of chymotrypsin soluble oxalic acid The stability of decarboxylase (" Sol "), oxalate decarboxylase crystal (" XTAL ") and crosslinked oxalate decarboxylase crystal (" CLEC ") Figure.

Figure 10 be describe to be spaced at the appointed time, in pH6.8, it is soluble in the presence of the simulated intestinal fluid containing pancreatin The stabilization of oxalate decarboxylase (" Sol "), oxalate decarboxylase crystal (" XTAL ") and crosslinked oxalate decarboxylase crystal (" CLEC ") The figure of property.

Detailed description of the invention

The present invention is based in part on following discoveries, can mitigate the height of mammal using oxalate decarboxylase (OXDC) crystal Oxaluria symptom.This document describes application OXDC crystal to treat the method for different oxalates associated conditions.Additionally, it is provided OXDC crystal and crosslinked crystal (CLECs) include and using their compositions.In addition, it discloses from prokaryotic host cell Cell extract in produce the methods of a large amount of albumin crystals.

Definition.In order to which the present invention is more easily understood, some terms are defined first.Other definition are illustrated in detailed description.

" biological sample " used herein is the biomaterial collected from cell, tissue, organ or organism, for example, with In detection and analysis object.Illustrative biological sample includes liquid, cell or tissue sample.Biological fluids include, for example, serum, blood Liquid, blood plasma, saliva, urine or sweat.Cell or tissue sample include biopsy, tissue, cell suspending liquid, or other samples and Sample, such as clinical sample.

" crystal " is a kind of form of solid matter, the atom (ginseng arranged it includes the pattern repeated with three-dimensional periodic See, for example, Barret, Structure of Metals, 2^nded.,McGraw-Hill,New York (1952)).Polypeptide Crystal form, for example, different from second of form-amorphous solid.Crystal shows unique feature, including shape, lattice Structure, percentage of solvents and optical property, for example, refractive index.

" device outside " is structure not in vivo, in the treatment of individual body fluid being made to contact OXDC crystal.It is preferred that Ground, device outside are the devices for the dialysis including Rend dialysis, for the device of continuous arteriovenous hemofiltration, in vitro Membrane oxygenator or for from other devices of blood flow filtering waste.Similarly, which includes the group of the device of filtering waste Part, including such as conduit, porous material or film.More specifically, device outside can be dialysis apparatus.It can also be dialysis apparatus Film.

" function fragment " of OXDC is a part for the OXDC polypeptides for the one or more bioactivity for remaining OXDC, institute State the ability that activity is for example catalyzed the decarboxylation of oxalates.Function fragment used herein can be included from one or two end End butt, unless otherwise indicated.It is saved for example, function fragment can have from the amino and/or carboxyl terminal of OXDC polypeptides Slightly 1,2,4,5,6,8,10,12,15, or 20 or more residues.Preferably, butt is no more than 20 from one or two end A amino acid.Function fragment can optionally be connected to one or more heterologous sequences.

Term " individual " or " subject " refer to arbitrary mammal, include but not limited to, any animal so classified, Including people, non-human primate, primate, baboon, orangutan, monkey, rodent (for example, mouse, rat), rabbit Son, cat, dog, horse, ox, sheep, goat, pig, etc..

Term " separated " refers to the molecule for substantially departing from its natural surroundings.For example, separated albumen is substantially de- From cell material or other albumen in the cell or tissue source from its source.The term refers to the purity of wherein separated albumen It is enough the product as therapeutic combination application,

Or at least 70% to 80% (w/w) purity, more preferably at least 80% to 90% (w/w) purity, more preferably 90 to 95% Purity；Most preferably at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8% or 100% (w/w) purity.

Terms used herein " about " refers to value ± 10% that up to term limits.For example, about 50mM refer to 50mM ± 5mM;About 4% refers to 4% ± 0.4%.

" oxalates associated conditions " used herein refer to the disease or disease related with the Pathological levels of oxalic acid or oxalates Disease, including but not limited to, hyperoxaluria, primary hyperoxaluria, intestines originality hyperoxaluria, idiopathic hyperoxaluria, ethylene glycol (oxalates) is poisoned, idiopathic urinary stone disease, kidney failure (including gradual, chronic or end stage renal disease), stearrhea, inhales Receive bad, ileum disease, Vulvodynia, cardiac conduction defects（cardiac conductance disorders）, inflammatory bowel disease, capsule Property fibrotic disease, pancreatic exocrine insufficiency, Crohn disease, ulcerative colitis, nephrocalcinosis, lithangiuria, and Kidney stone.Such illness and obstacle can optionally be acute or chronic.Known in the art and kidney, bone, liver, gastrointestinal tract The oxalates associated conditions related with pancreas.Additionally, it is also well known that calcium oxalate can deposit in Various Tissues, including, but not It is limited to, eyes, blood vessel, joint, bone, muscle, heart and the other vitals for causing many oxalates associated conditions.

In urine and the pH (pK of intestinal juice_a1=1.23,pK_a2=4.19), " oxalic acid " mainly using its salt form oxalates (as The salt of corresponding conjugate base) exist.Earnest,Adv.Internal Medicine24:407427(1979).Term " oxalic acid " It is used interchangeably in this disclosure with " oxalates ".Oxalates comprising lithium, sodium, potassium and iron (II) is soluble, but careless Sour calcium is usually very difficultly soluble in water (for example, being only dissolved to about 0.58mg/100ml at 18 DEG C.Earnest,Adv.Internal Medicine24:407427(1979)).Oxalic acid from food is also referred to as diet oxalates.The oxalic acid generated by metabolic process Salt is referred to as endogenous oxalates.It is the oxalates being present in instrument for circulation of body fluid such as blood to cycle oxalates.

Term " treatment effective dose " or " therapeutically effective amount " refer to, cause oxalates associated conditions（Including hyperoxaluria, Such as primary hyperoxaluria or intestines originality hyperoxaluria）Prevention, the delay of paresthesia epilepsy or the improved compound of symptom Amount.Therapeutically effective amount will be enough the seriousness for for example treating, preventing, mitigating the illness related with raised concentration of oxalate, Postpone its breaking-out and/or reduce the breaking-out danger of one or more symptoms.By well-known in the art and this specification Method described in further part, it may be determined that effective quantity.

Term " treatment ", " therapy " and their near synonym refer to the treatment and/or prevention of existing illness （prophylatic）/ prevention（preventative）Measure.Object in need for the treatment of can include having had certain medical disease In the individual of disease and danger in the illness or with the illness or may final diseased object.Following evaluation To the demand for the treatment of：For example, by the presence of the related one or more risk factors of the development with disease, the presence of illness or Progress or the possibility acceptance for the treatment of to the subject with the illness.Treatment can include slowing down or reversing illness Progress.

Oxalate decarboxylase.Oxalate decarboxylase (OXDC) (EC4.1.1.2) used herein refers to oxalic acid carboxy-lyase.Grass Acid decarboxylase is known in the art to be catalyzed oxalic acid according to following reactions independently of molecular oxygen (O2) and be oxidized to carbon dioxide With one group of enzyme of formic acid:

HO₂C-CO₂H→1CO₂+HCOOH

The isotype of oxalate decarboxylase and the sugared shape of those isotypes are included in this definition.The term is included from plant Object, bacterium and the OXDC of fungi, including the true oxalate decarboxylase from bacterium and fungi, such as bacillus subtilis, needle mushroom Or flammulina velutipes, aspergillus niger, pseudomonas, cyanobacteria category（Synechocystis sp.）, streptococcus mutans, Trametes trogii （Trametes hirsute）, sclerotinite, whiterot fungi（T.versicolor）, brown rot fungus（Postia placenta）, wart spore paint Plaque, Agaricus bisporus, methylotrophic bacteria（Methylobacterium extorquens）,Pseudomonas Oxalaticus, Ralstonia eutropha, Cupriavidus oxalaticus, Wautersia sp., Oxalicibacterium flavum,Ammoniiphilus oxalaticus,Vibrio oxalaticus,A.oxalativorans,Variovorax Paradoxus, xanthobacter autotrophicus, Eurotium, Penicillium, and Mucor.Optionally, OXDC can be additionally depended on Coacetylase, such as the OXDC from enteron aisle biology.In some cases, OXDC is soluble six glycoprotein polyprotein precursor.

Oxalate decarboxylase is generated by higher plant, bacterium and fungi, and with oxalic acid carboxy-lyase activity.Oxalic acid decarboxylation Enzyme includes those by following biology generations：Bacillus subtilis, needle mushroom or flammulina velutipes, aspergillus niger, pseudomonas, Cyanobacteria category（Synechocystis sp.）, streptococcus mutans, Trametes trogii（Trametes hirsute）, sclerotinite, whiterot fungi （T.versicolor）, brown rot fungus（Postia placenta）, myrothecium verrucaria, Agaricus bisporus, methylotrophic bacteria （Methylobacterium extorquens）, Pseudomonas oxalaticus, Ralstonia eutropha, Cupriavidus oxalaticus,Wautersia sp.,Oxalicibacterium flavum,Ammoniiphilus oxalaticus, Vibrio oxalaticus, A.oxalativorans, Variovorax paradoxus, xanthobacter autotrophicus, Eurotium, Penicillium and Mucor, they usually differentiate as cupin types OXDC.Cupin- sample albumen is that a major class shares some knots The albumen of structure feature.OXDC, such as it is active that the G-OXDC belonged to from Collybia, which is used as such as six polymer glycoproteins,.

Be used to prepare crystal and for the oxalate decarboxylase in methods described herein can from such as natural origin separate or Natural origin can be derived from.Terms used herein " being derived from " refers to there is naturally occurring amino acid or nucleic acid sequence in source Row.For example, the oxalate decarboxylase from bacillus subtilis includes the basic sequence of bacillus subtilis oxalate decarboxylase albumen, Or it is encoded by the nucleic acid for the sequence for being included in the coding oxalate decarboxylase found in bacillus subtilis or its degradation product.From one The albumen or nucleic acid in a source are included from the source is separated, recombinant production and/or chemical synthesis or modification molecule.This The crystal that text provides can be from the more of the OXDC function fragments of the amino acid sequence comprising OXDC or reservation oxalate degrading activity It is prepared by peptide.Preferably, OXDC retains at least one functional character of naturally occurring OXDC, for example, retaining following one or more It is multiple：It is catalyzed ability, multimerization ability and/or the manganese demand of oxalate degradation.

Separated oxalate decarboxylase.Oxalate decarboxylase has been isolated in the past, thus can have been obtained from many sources, including Bacillus subtilis, needle mushroom or flammulina velutipes, aspergillus niger, pseudomonas, cyanobacteria category（Synechocystis sp.）, become Shape streptococcus, Trametes trogii（Trametes hirsute）, sclerotinite, whiterot fungi（T.versicolor）, brown rot fungus（Postia placenta）, myrothecium verrucaria, Agaricus bisporus, methylotrophic bacteria（Methylobacterium extorquens）, Pseudomonas oxalaticus, Ralstonia eutropha, Cupriavidus oxalaticus, Wautersia sp., Oxalicibacterium flavum,Ammoniiphilus oxalaticus,Vibrio oxalaticus, A.oxalativorans, Variovorax paradoxus, xanthobacter autotrophicus, Eurotium, Penicillium, and mucor Belong to.OXDC can also be purchased from commercial supplier, for example, Sigma.It was once described from the method for natural origin separation OXDC, example Such as, in following bibliography:Tanner et al., The Journal of Biological Chemistry.47:43627- 43634.(2001);Dashek,W.V.and Micales,J.A.,Methods in plant biochemistry and molecular biology.Boca Raton,FL:CRC Press.5:49-71.(1997);Magro et al., FEMS Microbiology Letters.49:49-52.(1988);Anand et al., Biochemistry.41:7659-7669. (2002);and Tanner and Bornemann,S.Journal of Bacteriology.182:5271-5273 (2000).These separated oxalate decarboxylases can be used for forming crystal as described herein and method.

The oxalate decarboxylase of restructuring.Alternatively, the OXDC of restructuring can be used for being formed provided herein is crystal and method.Having In the case of a little, the OXDC of restructuring includes the sequence from naturally occurring OXDC sequences or is encoded by it.In addition, it is described herein Include the OXDC with naturally occurring sequence homology or substantially the same amino acid sequence.It also provides by with naturally depositing OXDC- homologous coding nucleic acids or substantially the same nucleic acid coding OXDC, and can crystallize as described herein, And/or application.

Herein referred to as " restructuring " polypeptide is the polypeptide generated by recombinant DNA method, is relied on including passing through Operate what is generated into the method for carrier in artificial recombination such as PCR (PCR) and/or using limitation enzyme clone Those." restructuring " polypeptide is also the polypeptide with the expression changed, such as in cell（Such as host cell）In have restructuring The naturally occurring polypeptide of the expression of modification.

In one embodiment, OXDC is from the core with bacillus subtilis or needle mushroom OXDC nucleic acid sequence homologous Sour recombinant production, and modified sometimes, for example, to increase or optimize the recombinant production in heterologous host.In SEQ ID NO:An example of the sequence of this modification is provided in 1 (nucleic acid), it includes the nucleic acid of the opening code-reading frame of needle mushroom OXDC Sequence, for being expressed in Candida boidinii.The OXDC sequences have modified to reduce its G/C content, it is connected To α mating factor secretory signal sequences, and the restriction endonuclease cleavage site of side joint engineering.In another embodiment party In case, OXDC is from SEQ ID NO:2 or can be in GenBank Accession No:The unmodified withered grass bud that Z99120 is obtained Spore bacillus OXDC nucleotide sequence recombinant productions.By SEQ ID NO:The amino acid sequence of 2 codings is provided as SEQ ID NO:3.

Can be used to form the OXDC polypeptides of OXDC crystal can express in host cell, such as include nucleic acid construct Host cell, the nucleic acid construct contain OXDC polypeptides or the coded sequence of its function fragment.It is suitble to the host of expression OXDC Cell can be yeast, bacterium, fungi, insect, plant or mammalian cell, for example, or genetically modified plants, transgenosis move Object or acellular system.Preferably, host cell can glycosylate OXDC polypeptides if necessary, can form disulfide bond, can secrete OXDC, and/or can support the multimerization of OXDC polypeptides.Preferred host cell includes but is not limited to, and Escherichia coli are (including big Enterobacteria Origami B and e. coli bl21), pichia pastoris, saccharomyces cerevisiae, schizosaccharomyces pombe, bacillus subtilis Bacterium, Aspergillus, Sf9 cells, Chinese hamster ovary (CHO), 293 cells (human embryo kidney), and other people's cells.Transgenosis is planted Object, transgenic animals（Including pig, ox, goat, horse, chicken and rabbit）And it is suitble to the host of production OXDC.

For recombinant production OXDC, host or host cell should include construct, and the latter is comprising at least one coding Plasmid, carrier, phasmid or the form of transcription or expression cassette of the nucleic acid of OXDC or its function fragment.It can obtain many kinds of structures Body, including the construct in the construct maintained with single copy or multiple copies or incorporation host cell chromosome.It is many heavy Group expression system, component and recombination expression reagent can be available commercially, such as from Invitrogen Corporation (Carlsbad,CA);U.S.Biological(Swampscott,MA);BD Biosciences Pharmingen (San Diego,CA);Novagen (Madison,WI);Stratagene (La Jolla,CA);and Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ),(Braunschweig,Germany)。

The recombination expression of OXDC optionally by allogeneic promoter, is controlled including composing type and/or inducible promoter.Start Son is for example, T7, alcohol oxidase (AOX) promoter, dihydroxy-acetone synthase (DAS) promoter, Gal1,10 promoters, phosphoric acid are sweet Oleic acid kinase promoter, glyceraldehyde-3-phosphate dehydrogenase promoter, alcohol dehydrogenase promoter, copper metal sulfoprotein (CUP1) open Mover, acid phosphatase promoter, CMV and promoter polyhedrin are also suitable.Based on host or host cell, selection Specific promoter.It in addition, for example, can be by methanol, copper sulphate, galactolipin, hypophosphate, alcohol（For example, ethyl alcohol）Induction is opened Mover can also use, and be well-known in the art.

The nucleic acid of coding OXDC can optionally include heterologous sequence.For example, in some embodiments, by secretion sequence Included in the N- ends of OXDC polypeptides.Signal sequence can be used, such as from α mating factors, BGL2, yeast acid phosphatase (PHO), those of zytase, alpha amylase, the albumen from other yeast secretaries those and from host can be instructed thin The secreting signal peptide of other species of the secretion of born of the same parents.Similarly, other heterologous sequences such as attachment is (for example, it includes cuttings Or restriction endonuclease site) and one or more expression control elements, enhancer, terminator, leader and one Or multiple translation signals, all in the range of the description.These sequences can be optionally included in construct and/or be connected to On the nucleic acid for encoding OXDC.Unless otherwise indicated, " connection " sequence can be combined directly to each other or indirectly.

Similarly, epitope or affinity tag such as histidine, HA (hemagglutinin peptide), maltose-binding protein,FLAG or glutathione-S-transferase can be optionally coupled on OXDC polypeptides.In production or after purification, Can optionally lower label be cut from OXDC.Technical staff can be readily selected suitable heterologous sequence, for example, matching place Chief cell, construct, promoter, and/or secretory signal sequence.

OXDC homologues or variant differ one or more residues with OXDC canonical sequences.It such as can be with similar in structure Amino acid replacement some amino acid for specifying.Similar amino acid includes in structure:(I, L and V);(F and Y);(K and R);(Q And N);(D and E);(G and A).OXDC homologues as described herein also include missing, addition or the displacement of amino acid.It is such Homologue and variant include：(i) polymorphie variant and natural or artificial mutant, the polypeptide of (ii) modification, wherein modification One or more residues, and (iii) include the mutant of the residue of one or more modifications.

If it has at least 40% with canonical sequence, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% homogeneity, then OXDC polypeptides or nucleic acid are " homologous " (or " homologues ").If homologue with reference to sequence Row are different, then it is " variant ".If the nucleotide or amino acid sequence of homologue are differed with canonical sequence (for example, by cutting Short, missing, displacement or addition) it is no more than 1,2,3,4,5,8,10,20 or 50 residue and retains the energy of catalysis oxalate degradation Power (or coding retain the ability polypeptide), then homologue with reference to OXDC sequences " substantially the same ".The piece of oxalate decarboxylase Section can be homologue, including variant and/or substantially the same sequence.As example, homologue can be derived from different OXDC sources or they can have by truncating, lacking, replacing or adding mutation from canonical sequence or with canonical sequence It closes.2 homogeneity percentages between nucleotide or amino acid sequence can be measured by the alignment algorithm of standard, for example, In Altschul et al., J.Mol.Biol.,215:Basic Local Alignment Tool described in 403410 (1990) (BLAST), Needleman et al., J.Mol.Biol.,48:Algorithm or Meyers of 444453 (1970) et al., Comput.Appl.Biosci.4:The algorithm of 1117 (1988).Such algorithm mixes BLASTN, BLASTP, and (summary is shown in McGinnis and Madden, Nucleic Acids Res. to " BLAST2Sequences " program32:W20-W25, 2004).When using such program, default parameters can be used.For example, for nucleotide sequence, it can be by following settings For " BLAST2Sequences ":Program BLASTN matches 2 rewards（reward for match2）, 2 point penalties of mispairing （penalty for mismatch 2）, room opens（open gap）And gap extension penalties（extension gap penalties）It is 5 and 2 respectively, gap x_dropoff50, expect10, word size 11, filter ON.For amino Acid sequence, can be by following settings for " BLAST2Sequences ":Program BLASTP, matrix B LOSUM62, room open （open gap）And gap extension penalties（extension gap penalties）It is 11 and 1 respectively, gap x_dropoff 50,expect 10,word size 3,filter ON.Suitably form the amino acid of the OXDC of crystal described herein and nucleic acid sequence Row can include homologous, variant or substantially the same sequence.

The purifying of oxalate decarboxylase.It, can be from source before crystallization（Such as natural or restructuring source）Purifying Oxalate decarboxylase albumen or polypeptide.Herein referred to as " separated " polypeptide is substantially to depart from its natural surroundings（For example, They origin (for example, cell, organize (that is, plant tissue) albumen, lipid, and/or nucleic acid or liquid or culture medium ( In the case of secrete polypeptide))）Polypeptide.Separated polypeptide includes what is obtained by methods described herein or other appropriate methods Those, including substantially pure or substantially pure polypeptide and by chemical synthesis, by recombinant production or by biology and The polypeptide of the combination production of chemical method.Optionally, separated albumen passes through be processed further after manufacture, such as passes through purifying Step.

Purifying can include buffering fluid exchange and chromatographic step.It is optionally possible to using concentration step, for example, by saturating Analysis, chromatofocusing chromatography, and/or combination buffer are replaced.In some cases, cation or anion-exchange chromatography are used for Purifying, including Q- agaroses, DEAE agaroses, DE52, sulfopropyl Sepharose Chromatography or CM52 or similar cation exchange column. Fluid exchange is buffered optionally before chromatographic isolation, and can for example be percolated to realize by grossflow filtration.In some products In, OXDC is the purity of at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9%.

The purifying carried out with gram quantity grade is suitable for preparing OXDC, for effective, cheap, production scale OXDC purifying optimizations Operation.For example, at least 0.5,1,2,5,10,20,50,100,500 or 1000 gram or more of OXDC is provided in purification process Purifying.In an illustrative operation, the initial sample for providing at least 10L, 50L, 100L, 500L, 1000L or more is cut Line flows through filter, to realize the precipitation of buffering fluid exchange and contaminating protein.Single Q- agarose columns are optionally for purifying OXDC.

The crystallization of the OXDC of purifying can also remove pollutant, such as OXDC products are further purified.For example, with it is soluble The OXDC of purifying is compared, and the OXDC crystallized as described in embodiment 2-6, which has, drops low-level low molecular weight pollutant.At some It is (logical selectively to exclude the measurement quality with 0-10KDa, 1-10KDa, 0.5-5KDa or 2-5KDa from crystal form for aspect Cross the laser desorption ionisation mass spectral analysis (MALDI-MS) of Matrix-assisted) pollutant.For example, by crystallization, it can be basic The pollutant of upper measurement quality of the removal with about 2.5,3.0,3.7,3.8,4.0,4.2 or 5.0KDa.Using for example containing thick The fermentation medium of oxalate decarboxylase, can also crystallization purifying.

The crystallization of oxalate decarboxylase.Using above-mentioned OXDC polypeptides such as six aggressiveness, oxalate decarboxylase crystal can be prepared (referring to Anand et al., Biochemistry41:7659-7669(2002)).Such as steam diffusion can be used (for example, hanging drop With sitting drop method) and batch crystallization method.By making controlled crystallization in aqueous solution of the albumen from aqueous solution or comprising organic solvent, Oxalate decarboxylase crystal can be gradually formed.The evaporation rate of the condition that can be controlled including such as solvent, appropriate cosolute and Presence, pH and the temperature of buffer.

It is applied for treatment, for example, it is big for treating the illness related with levels of oxalate or obstacle, a variety of OXDC crystal Small is suitable.In certain embodiments, using the crystal less than about 500 μm of average-sizes.Also provide with about 0.01, 0.1st, the average of 1,5,10,25,50,100,200,300,400,500 or 1000 μm of length, maximum or minimum dimension be (for example) Oxalate decarboxylase crystal.Crystallite showers is also suitable.

Scope is suitable, and technical staff would appreciate that.For example, albumin crystal can have about 0.01 μm to about 500 μm, Or 0.1 μm to about 50 μm of longest dimension.In a specific embodiment, longest dimension scope is about 0.1 μm to about 10 μ m.Crystal can also have selected from ball, pin, stick, the shape of plate, such as hexagon and square, diamond shape, cube, bipyramid and Prism.In explanatory embodiment, crystal is the cube with the longest dimension less than 5 μm.

In general, by the albumen that will crystallize with appropriate aqueous solvent or containing appropriate crystallizing agent（Such as salt or have Solvent）Aqueous solvent it is combined, produce crystal.Make the solvent and albumen combined, and optionally fitted in measuring It closes induction crystallization and maintains protein active and the acceptable temperature experience stirring of stability.Solvent can optionally include molten altogether Matter, such as unit price or bivalent cation, co-factor or chaotropic agent and the buffer for controlling pH.Measuring promotes crystallization institute The cosolute needed and their concentration.During commercial scale, for example, by making albumen, precipitating reagent in batch process, being total to Solute and optional buffering Solution-Phase Combinatorial can carry out generating the controlled precipitation of crystallization.Alternative laboratory knot can be adopted Crystal method and condition, for example, dialysis or steam diffusion (McPherson, et al., Methods Enzymol.114:112-20 (1985)and Gilliland,Crystal Growth90:51-59(1998)).Once in a while, between crosslinking agent and crystallization medium Incompatibility may need to change buffer solution (solvent) before crosslinking.

As described embodiments, oxalate decarboxylase can crystallize under a number of conditions, including wide pH scopes (for example, pH3.5- 8.0).In some described embodiments comprising precipitating reagent such as polyethylene glycol (for example, PEG200, PEG400, PEG600, PEG1000, PEG2000, PEG3000, PEG8000 [referring to embodiment 7 and 8]) or organic cosolvent such as 2- methyl -2,4- penta Glycol (MPD).The ordinary salt that can be used includes sodium chloride, potassium chloride, ammonium sulfate, zinc acetate etc..

Concentration of the oxalate decarboxylase in zymotic fluid is crystallized can be, for example, at least 5,10,15,20,25,30,35,40, 45,50,60,70,80,90, or 100mg/ml, or higher.The efficiency or yield of crystallization reaction are at least 50%, 60%, 70%, 80%th, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher.In one embodiment, by batches Process makes oxalate decarboxylase solution be mixed with appropriate buffer solution, gradually forms or produce oxalate decarboxylase crystal.Some In embodiment, the buffer solution is 100mMTris-HCl pH of buffer 8.0 and containing 2mM cysteines-HCl 100mMNaCl。

It is crystallized from cell or cell extract.Crystal can be prepared directly from cell or crude cell extract.In a reality It applies in scheme, the bacterial cell of harvest expression oxalate decarboxylase.The suspension cell, and even again in the presence of DNA enzymatic is with or without Pulp.Salting liquid is added in into cell lysate, reaches about 0.3M, 0.4M, 0.5M, 0.6M or higher salinity.The salt of addition Can be sodium salt, sylvite, calcium salt or other salt.By removing cell fragment, optionally egg can be extracted from cell mixture In vain.In one embodiment, the cell mixture of homogenization is centrifuged, makes albumen in supernatant soln.It is thin by reducing The salinity of born of the same parents' mixture or protein solution generates crystal.In one embodiment, desalination is gone by dialysis, to maintain egg White concentration.In order to improve crystal yield, can reduce solution salinity before protein concentrate solution.It can be in pH about 6,7 Or 8 solution in generate crystal.

It can be from albumen precipitation object or particle preparation crystal.In one embodiment, harvest expression target protein is thin Born of the same parents, and oxalate decarboxylase albumen is collected in sediment or particle.Particle containing oxalate decarboxylase albumen or sediment is molten In salting liquid.By reducing the salinity of protein solution, crystal is formed.In order to improve crystal yield, produced reducing concentration Before crystal, the salinity in the protein solution of dissolving is at least about 0.3M, 0.4M, 0.5M or higher.

Crystal can also be prepared from protein solution.In one embodiment, oxalate decarboxylase egg is concentrated in salting liquid White solution when reducing the salinity of solution, forms crystal.In order to improve crystal yield, reduce concentration come produce crystal it Before, salinity is at least about 0.3M, 0.4M, 0.5M or higher.

Stabilized crystal.Once oxalate decarboxylase crystal gradually forms in suitable medium, can be optionally Them are stabilized, such as passes through crosslinking.By introducing covalent bond between the component protein molecule of crystal, crosslinking causes lattice Stabilisation.This causes protein delivery to otherwise may there are incompatible or even deposited with intact proteins with the lattice It is possibly realized in incompatible alternative environment.Oxalate decarboxylase crystal can be for example, by amine groups, sulfydryl (sulphur hydrogen Base) and carbohydrate portions be crosslinked.Crosslinked crystal be also referred to as herein " OXDC-CLEC, " " CLEC-OXDC, " or “CLEC”。

Crosslinked crystal can change enzyme stability (for example, pH, temperature, mechanically and/or chemically stability), OXDC activity PH properties, solubility, the homogeneity of crystal size or volume, the rate that enzyme is discharged from crystal, and/or in basic lattice Hole size and shape between each enzyme molecule.

Advantageously, so that crystal include show at least 60% compared with soluble OXDC, 80%, 100%, 150%, 200%, 250%th, the mode of 300% or higher active OXDC, is crosslinked or is stabilized according to the present invention.Compared with soluble OXDC, Stability can improve at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300% or more It is more.Stability, such as pH stability, temperature stability, the stability of anti-erepsin, dissolution can be measured under condition of storage Stability and vivo biodistribution stability, such as.

In some embodiments, crosslinking can slow down dissolution of the OXDC polypeptides into solution in crystal, effectively by egg White molecule is immobilized in microcrystal grain.After triggering agent in the ambient enviroment exposed to crosslinked albumin crystal, such as Using rather than condition of storage under, protein molecular can slow mechanism dissolved, release active OXDC polypeptides and/or increase OXDC activity. Dissolution rate can be controlled, such as passes through one or more of following factors:The degree of cross linking, albumin crystal is exposed to crosslinking agent Time span adds in the rate of crosslinking agent, the property of crosslinking agent, the chain length of crosslinking agent, pH, temperature, thiol into albumin crystal （sulfahydryl）Reagent（Such as cysteine, glutathione）Presence, the surface area of crosslinked albumin crystal, crosslinked egg The size of Bai Jingti, and the shape of crosslinked albumin crystal.

Be crosslinked (parallelly) or sequentially to be realized using one kind in a variety of crosslinking agents or combination thereof at the same time, Including multi-functional dose.It is and such multi-functional after the triggering agent in ambient enviroment or by given time phase Crosslinking between the albumin crystal of cross-linking agents becomes smaller or dies down, and causes albumen dissolution or activity release.Alternatively, crosslinking can be with It is broken in binding site, causes albumen dissolution or activity release.Referring to U.S. Patent number 5,976,529 and 6,140,475.

In some embodiments, crosslinking agent is that have at least 2,3,4,5, or more active part multi-functional crosslinking Agent.In different implementation scenarios, the reagent can be selected from glutaraldehyde, butanedial, suberic aldehyde, glyoxal, the double (ambers of two sulphur Imide propionic ester), bis- sulphur of 3,3' is double (thiosuccimide base propionic ester), 3,3 '-two thiobis the third imidic acid diformazans Ester HCl, N- succinimido -3- (two sulphur of 2- pyridyl groups) propionic ester, cyclohexanediamine, diamino-octane, ethylenediamine, amber Acid anhydrides, phenyl glutaric anhydride, salicylide, acetimide ester（acetimidate）, formalin, methacrylaldehyde, amber semialdehyde, fourth Aldehyde, lauryl aldehyde, glyceraldehyde, and trans-oct-2-ene aldehyde.

Other polyfunctional crosslinking agent include halogenated triazine, for example, cyanuric chloride;Halogenated pyrimidine, for example, 2,4,6- trichlorines/ Bromopyrimidine;Aliphatic series or the acid anhydrides or halide of aromatic monocarboxylate or dicarboxylic acids, for example, maleic anhydride, (methyl) acryloyl chloride, chlorine Chloroacetic chloride;N- methylol compounds, for example, the chloro- acetamide of N- methylols;Diisocyanate or diisothio-cyanate, for example, sub- Phenyl -1,4- diisocyanate and aziridine.Other crosslinking agents include epoxides, for example, dicyclic oxide, three epoxidations Object and four epoxide.In one embodiment, crosslinking agent is glutaraldehyde, i.e., difunctional dose a kind of, and glutaraldehyde is used alone Or it is used with epoxides order.It can also be with reversible cross-linking agent（Example it is described as follows those）It (parallelly) or sequentially uses simultaneously Other crosslinking agents (see, e.g., the catalogues in 1996 of Pierce Chemical Company).

According to an alternative embodiment of the present invention, can parallelly or sequentially be handed over using reversible cross-linking agent Connection.Obtained crosslinked albumin crystal is characterized in that the multi-functional cross-linking agent of reactivity, and wherein trigger is mixed as separating group Enter.Reactive functionality participation connects together the reactive amino acid side chain in albumen, and trigger is by that can pass through change week One or more of collarette border condition (for example, pH, the presence of reducing agent, temperature or thermodynamics water activity) is come the key that destroys Composition.

Crosslinking agent can be congenerous or exclusive-OR function.Reactive functionality (or part) can be with for example, selected from following officials One of can roll into a ball (wherein R, R ', R ", and R ' ' ' they can be alkyl, aryl or hydrogen group):

I. reactive acry radical donor, such as:Carboxylate RCOOR ', amide RCONHR ', acyl azide RCON₃, carbodiimide R- N=C=N-R ', N hydroxyl imide ester, RCO-O-NR ', imines ester R-C=NH2⁺(OR '), acid anhydrides RCO-O-COR ', carbonic ester RO- CO-O-R ', urethanes RNHCONHR ', acid halide RCOHal (wherein Hal=halogen), acid hydrazide RCONNR ' R ", and （O- acyl isoureas）OAcylisoureas RCO-O-C=NR’(-NR”R’’’)；

II. reactive carbonyl, such as:Aldehyde RCHO and ketone RCOR ', acetal RCO (H₂) R ', and ketal RR ' CO2R ' R " ( Document (Pierce Catalog and Handbook, Pierce Chemical Company, Rockford, Ill. (1994); S.S.Wong,Chemistry of Protein Conjugation and Cross-linking,CRC Press,Boca Protein immobilization is described in Raton, Fla. (1991) and is crosslinked known to the technical staff in field containing reactive carbonyl Functional group)；

III. alkyl or aryl donor, such as:Alkyl or aryl halide R-Hal, azide R-N₃, sulfuric ester RSO₃R ', phosphate RPO (OR '₃), alkyl oxygenSalt R₃O+, sulfonium R₃S+, nitrate RONO₂, Michael receptors RCR '=CR ' ' ' COR ", aryl fluoride ArF, isonitrile RN+=C-, halogenated amine R₂N-Hal, alkene, and alkynes;

IV. sulfur-containing group, such as:Disulphide RSSR ', sulfydryl RSH, and epoxides R₂C_^OCR’₂;With

V. salt, such as:Alkyl or aryl ammonium salt R₄N+, carboxylate RCOO-, sulfate ROSO₃-, phosphate ROPO₃", and Amine R₃N。

Reversible crosslinking agent, for example, including trigger.Trigger include alkyl, aryl, or with can with it is crosslinked Other chains of the activated group of albumen reaction.Those reactive groups can be the group of any kind, such as be easy to nucleophilic and put Change, free radical displacement or electrophilic displacement those, including halide, aldehyde, carbonic ester, urethanes, xanthane and ring Oxide and other.For example, reactive group can be to acid, alkali, fluoride, enzyme, reduction, oxidation, sulfydryl, metal, light point Solution, root（radical）Or thermally labile.

In T.W.Green, Protective Groups in Organic Synthesis, John Wiley ＆ Sons (Eds.) other examples of reversible crosslinking agent are described in (1981).It can will be for the plan of any kind of reversible protecting groups Slightly incorporation is suitble in the crosslinking agent of crosslinked protein crystal that production can realize reversible controlled dissolution.Different schemes are listed in The Angewante Chemie Inl.Ed.Engl. of Waldmann,35:2056 (1996) are in the summary of the theme.

Other types of reversible crosslinking agent is containing disulfide bond crosslinking agent.Crosslinking agent destruction in this way is cross-linked to form Trigger be that reducing agent, such as cysteine are added in into the environment of crosslinked albumin crystal.In Pierce Catalog and Illustrative disulphide crosslinking agent is described in Handbook (1994-1995).Disclosed in U.S. Patent number 6,541,606 The example of such crosslinking agent and method, relevant portion are incorporated by reference into.

Alternatively, it is also possible to use between carbohydrate portions or be crosslinked between carbohydrate portions and amino acid Crosslinking agent.

The concentration of cross-linking agent solution can be about 0.01% to 20%, about 0.02% to 10% or about 0.05% to 5%w/v.It is logical Often, crosslinking agent is about 0.5% or about 1%w/v.For example, the concentration of cross-linking agent solution can be, for example, about 0.01%, 0.02%, 0.05%th, 0.075%, 0.1%, 0.2%, 0.3%, 0.5%, 1%, 2%, 3%, 3.5%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15% or 20%w/v [referring to the table 2 in embodiment].It may need to replace buffer solution before crosslinking.Can optionally lyophilized or with Other manner prepares crystal, including CLEC.

Crystal including crosslinked crystal as described herein, can be used for therapy as described herein and reduce oxalates In horizontal method.OXDC crystal can be used for and industrial process (for example, synthesis, processing, bioreediation sterilize, sterilizing) Related method and treatment plant（Such as mycotic infection of plant）Method, such as summarize see, e.g., Svedruzic etc. People, Arch.Biochem.Biophys.433:176-192(2005).Such non-treatment of soluble or unbodied OXDC Purposes is see, e.g., U.S. Patent number 5,866,778;6,218,134;6,229,065;6,235,530;With 6,503,507. One or more properties of OXDC crystal based on aforementioned stable, crystal as described herein can be used for these purposes, such as Increase the stability of oxalate decarboxylase.

The drying of oxalate decarboxylase crystal.Water removal, organic solvent or liquid polymers are gone by following manner, so as to dry Oxalate decarboxylase crystal:Including being dried with nitrogen, air or inert gas, vacuum drying oven drying, lyophilized uses volatility Organic solvent washing, then evaporate solvent, evaporated in draught cupboard, tray drying, fluidized bed drying, spray drying, vacuum Dry or roller drying.In general, when crystal becomes free flowable powder, it is dried.It is wet by passing through gas stream Crystal can be dried.The gas can be selected from:Nitrogen, argon, helium, carbon dioxide, air or combination thereof.

In principle, it is possible to dry crystal is prepared by lyophilized.But the technology includes the fast quickly cooling of substance But, it is only applicable to freeze the product of stabilization.In one embodiment, first by the aqueous solution containing lenticular oxalate decarboxylase - 40 to -50 DEG C are chilled to, is then taken out under vacuo.

The production of oxalate decarboxylase crystal or preparation or composition comprising such crystal.In one aspect, disclose Oxalate decarboxylase crystal or preparation or composition comprising such crystal.Such composition can be made according to following methods It is standby:

First, crystallize oxalate decarboxylase.Then, sugar, sugar alcohol, tackifier, wetting agent or solubilizer, buffering will be selected from Salt, emulsifier, antimicrobial, the excipient or ingredient of antioxidant and coating agent are directly added into mother liquor.Alternatively, removal is female Liquid, then by crystal suspension when excipient solution is minimum 1 hour small to most 24.Excipient concentration typically about 0.01 is to about 10%(w/w).The constituent concentration is about 0.01 to about 90% (w/w).The crystal concentration is about 0.01 to about 99% (w/w).

Then by filtering or by centrifugation, mother liquor is removed from crystal slurry.Then, optionally in room temperature or about -20 DEG C to about 25 DEG C of temperature, with one or more organic solvents（For example, ethyl alcohol, methanol, isopropanol or ethyl acetate）About 50- 100% (w/w) solution washs crystal.

Then by the way that nitrogen, air or inert gas flow is made to pass through them, dry crystal.Alternatively, by being air-dried, spraying Mist drying, lyophilized or vacuum drying, dry crystal.After washing, dry a minimum of about 1 hour to most about 72 it is small when, until The water content of final product is below about 10%（By weight）, more preferably less than about 5%（By weight）.It finally, if necessary can be with Carry out the macro of crystal（Reduce size）.

An embodiment according to the present invention, when prepare oxalate decarboxylase crystal or preparation comprising such crystal or During composition, reinforcing agent, such as surfactant are added without in crystallization process.The excipient or ingredient are added after crystallisation Enter in mother liquor, concentration is that either concentration is about 0.1 to about 25% (w/w) to about 1 to about 10% (w/w) or concentration is about 0.1 To about 50% (w/w).By the excipient or ingredient and the crystal in mother liquor incubate about 0.1 to about 3 it is small when or incubate about 0.1 To about 12 it is small when or incubate about 0.1 to about 24 it is small when.

In another embodiment of the present invention, the ingredient or excipient are dissolved in the solution beyond mother liquor, from Mother liquor takes out crystal, is suspended in excipient or ingredient solution.The ingredient or excipient concentration and incubative time with it is above-mentioned Those are identical.

It is a further advantage of the invention that it can be taken off by lyophilized to dry the oxalic acid being encapsulated in polymeric carrier Decarboxylase crystal or its preparation are to form the composition for including microballoon.Lyophilized or freeze-drying allow to isolate from composition Water.Oxalate decarboxylase crystalline composition is freezed first, is subsequently placed in high vacuum.In a vacuum, water sublimate is crystallized, is left oxalic acid Decarboxylase crystals composition, only containing the water combined closely.Such processing can be such that composition further stabilizes, and make The storage and transport of common environment temperature are easier.

Spray drying allows to separate water outlet from crystalline articles.It is highly suitable for the suspension from solution, emulsion and pumpable The drying solid of continuous production powder, particle or lump form in the liquid charging stock of liquid form.Spray drying includes, and liquid is former Material is atomized into droplet spray, and makes droplet in dry indoor exposures hot-air.Pass through rotary (wheeled) or nozzle spray device Generation spraying.Under the conditions of controlled air temperature and current, moisture is evaporated from droplet, forms dry particle.Spray-drying operation Need relatively high temperature.But be normally only slight to the pyrolytic damage of product, this is because in crucial drying stage Evaporation cooling effect and because dry matter be subsequently exposed to high temperature time it is very short.Powder is continuously arranged from hothouse Go out.According to the drying feature and powder specifics of product, selection operation condition and drier design.Spray drying is a kind of preferable Process, wherein final product must meet accurately in terms of particle size distribution, residual moisture content, heap density and grain shape Quality standard.

Composition.OXDC crystal, including crosslinked crystal, as composition such as pharmaceutical composition (see, e.g., U.S. State's patent No. 6,541,606, it describes the preparation and composition of albumin crystal) it provides.Include the medicine group of OXDC crystal Closing object includes OXDC crystal and one or more ingredients or excipient, includes, but are not limited to sugar and the polymer of bio-compatible.It assigns The example of shape agent is described in American Pharmaceutical Association（American Pharmaceutical Association）With pharmaceutical society of Britain （Pharmaceutical Society of Great Britain）The Handbook of Pharmaceutical of combined publication In Excipients, other examples are as described below.

OXDC enzymes can be as any one of a variety of physiologically acceptable salt forms as crystal in the composition To apply and/or by the use of acceptable pharmaceutical carrier and/or additive as a part for pharmaceutical composition.Physiologically it is subjected to Salt form and standard pharmaceutical compounding techniques and excipient be it is well known to the skilled person (see, e.g., Physician’s Desk Reference(PDR)2003,57th ed.,Medical Economics Company,2002; And Remington:The Science and Practice of Pharmacy compile .Gennado et al., 20th ed, Lippincott,Williams & Wilkins,2000).For application purpose, " preparation " includes " crystal formulations ".

It can be combined with excipient available for the oxalate decarboxylase in the method for the present invention.According to the present invention, " excipient " The filler combination used as filler or in pharmaceutical composition.For the illustrative ingredient and excipient in composition As described below.

The polymer of bio-compatible.The polymer of bio-compatible is non-antigenic (when being not used as adjuvant), non-cause It is cancer, nontoxic and additionally they be not with living organism inherently incompatible polymers, they can be used for this paper institutes In the OXDC crystalline compositions stated.Example includes:Poly- (acrylic acid), poly- (cyanoacrylate), poly- (amino acid), poly- (acid Acid anhydride), poly- (depsipeptide), poly- (ester) for example poly- (lactic acid) or PLA, poly- (lactide-co-glycolic) or PLGA, poly- (beta-hydroxy Butyrate), it is poly- (caprolactone) and poly- (dioxanone);Poly(ethylene glycol), poly- ((hydroxypropyl) isobutyl Methacrylamide, Poly- [（It is organic）Phosphine nitrile], poly- (adjacent ester), poly- (vinyl alcohol), poly(vinyl pyrrolidone), maleic anhydride-alkyl vinyl ether is common Polymers, pluronic polyols（pluronic polyols）, albumin, alginate esters, cellulose and cellulose derivative, Collagen, fibrin, gelatin, hyaluronic acid, oligosaccharides, glucosaminoglycan, sulfated polysaccharides, their admixture and copolymer.

Biodegradable polymeric, you can with the polymer degraded by hydrolyzing or dissolving, it may be embodied in OXDC In crystalline composition.Degradation can be (the equably entire polymerization of degradation of heterogeneous (occurring mainly in particle surface) or homogeneity Object matrix).

Can in OXDC crystalline compositions comprising such as one or more excipient or drug ingedient or excipient into Point.Ingredient can be inert or active ingredient.

With the method for OXDC crystal treatment oxalates associated conditions.The method of the present invention includes to mammalian subject Using oxalate decarboxylase, such as OXDC crystal or its cross-linked form, have to treat, prevent or reduce with raised levels of oxalate The illness of pass is caused danger.Raised levels of oxalate can be detected in the biological sample for example from subject, such as Body fluid, including urine, blood, serum or blood plasma.In certain embodiments, it is horizontal to detect urinary oxalate.In side as described herein In method, crystal and/or the crystalliferous composition of bag can be applied.

In some embodiments, treatment is provided with primary hyperoxaluria, intestines originality hyperoxaluria, surgical operation Caused by hyperoxaluria, idiopathic hyperoxaluria, oxalosis individual hyperoxaluria method.In other situations Under, kidney, bone, liver, the raised oxalates associated conditions of gastrointestinal tract and pancreas can be suitable for treatment disclosed herein.Pass through herein Other conditions or diseases of the method treatment of offer include but is not limited to, ethylene glycol (oxalates) poisoning, idiopathic lithangiuria Disease, kidney failure (including gradual, chronic or end stage renal disease), stearrhea, malabsorption, ileum disease, Vulvodynia, heart pass Lead obstacle（cardiac conductance disorders）, inflammatory bowel disease, cystic fibrosis, exocrine pancreas function is not Entirely, Crohn disease, ulcerative colitis, nephrocalcinosis, osteoporosis, lithangiuria, and kidney stone.Such illness Can optionally be acute or chronic with obstacle.

The method of the present invention can make the levels of oxalate in subject and the level in untreated or control subject Compared to reduction at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more.In some embodiments, lead to The levels of oxalate of comparison subject before and after application OXDC is crossed, measurement reduces.In some embodiments, it is of the invention It provides treatment or improves the method for oxalates associated conditions or obstacle, so that one or more symptoms of the illness or disorder Improve at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more.In certain embodiments, it is described Method reduces the level of endogenous oxalates and/or the absorption of diet oxalates.

In some embodiments, the side of individual of the treatment with the genotype related with high levels of oxalate is provided Method, such as reduce mutant homozygous or heterozygosis the individual of following enzymatic activitys：For example, alanine:Glyoxylate aminotransferase, second Aldehydic acid reductase/hydroxypyruvate reductase, liver oxyacetate oxidizing ferment or participation oxalic acid salt metabolism or related with hyperoxaluria Another enzyme.In other embodiments, treatment is provided with reducing or lack Oxalobacter formigenes intestines cluster The method of individual.

Disclosed method includes, to it is susceptible, with the illness related with raised levels of oxalate or in the danger Mammalian subject apply therapeutically effective amount oxalate decarboxylase.By the present invention method treat group include but It is not limited to, by oxalates associated conditions（For example, primary hyperoxaluria or intestines originality hyperoxaluria）Or in the development disease Subject in the danger of disease.

The subject of the method according to the invention treatment includes but is not limited to mammal, and including people, non-human primates move Object, primate, baboon, orangutan, monkey, rodent (for example, mouse, rat), rabbit, cat, dog, horse, ox are continuous Sheep, goat, pig, etc..

Indication, symptom, and disease indication.Many methods can be used for judge oxalates associated conditions or with raised grass The development of the horizontal related illness of hydrochlorate or progress.Such illness includes but is not limited to, as defined above arbitrary illness, disease Disease or obstacle.It, can for example, by measuring urinary oxalate, plasma oxalate salt, measuring kidney or liver function or detecting calcium oxalate deposits object To judge the development of oxalates associated conditions or progress.

By detecting or measuring the concentration of oxalate in such as urine sample or other biological samples or liquid, it is possible to authenticate disease Disease, disease or obstacle.The early symptom of hyperoxaluria is typically kidney stone, may be with serious or burst abdominal pain or the side of body Bitterly, blood urine, frequent urgent urination, urodynamic or fever and shiver with cold.Kidney stone can be Symptomatic or asymptomatic, and can be with It is observed for example, by abdomen X-ray, ultrasound or computerized tomography (CT) scanning imagery.If not controlling hyperoxaluria, kidney meeting It is impaired, and impaired renal function.Kidney possibly even failure.By the reduction of urinary output or lack (glomerular filtration rate), general Diseased sensation, tired and significant fatigue, Nausea and vomiting, anaemia and/or be difficult in the normal development of child's period and growth, It can differentiate kidney failure (and poor kidney).By directly estimating (such as with eyes), X-ray, ultrasound, CT, ultrasonic cardiography The methods of figure or biopsy (for example, bone, liver or kidney), can also detect the calcium oxalate deposits in other tissues and organ Object.

Using art-recognized directly or indirectly measuring method, kidney and liver function and concentration of oxalate can also be judged. By widely-known technique, compounds content or urine, blood or other biological samples can also be tested.For example, it can measure Oxalates, oxyacetate and glycerate are horizontal.The measuring method of liver and renal function is well-known, for example, analysis hepatic tissue Azymia and analysis nephridial tissue oxalate deposits object.Can also having notified for test sample cause primary hyperoxaluria DNA changes.

Other treatment indications include but is not limited to, there are one or more risk factors, including front and portion below Divide those discussed.By finding out risk factor as one or more, diagnosis or the presence or absence of prognostic indicator, Ke Yijian Other places in development or susceptible illness, disease or obstacle subject or may especially be easily accepted by oxalate decarboxylase treatment it is tested Person.Similarly, by analyzing one or more genotypes or phenotypic marker, it is possible to authenticate in development oxalates associated conditions Danger in individual.

The urinary oxalate level that disclosed method can be used for every 24 hours period is at least 30,40,50,60,70,80, 90、100、110、120、130、140、150、160、170、180、190、200、210、220、230、240、250、260、270、 280th, the subject of 290,300,310,320,330,340,350,360,370,380,390 or 400mg oxalates or more. In some embodiments, levels of oxalate is related with one or more symptoms or symptom.The oxalic acid in biological sample can be measured Salt level, such as body fluid, including blood, serum, blood plasma or urine.Optionally, oxalates is standardized into the albumen or object of standard Kreatinin in matter, such as urine.In some embodiments, it is desirable that the method for protection includes, using oxalate decarboxylase, 1,3, 5th, in 7,9,12 or 15 days, before being reduced to the level that can not be detected by the Xun Huan levels of oxalate of subject or be reduced to treatment Subject's levels of oxalate is less than 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70% or 80%.

The urinary oxalate excretion that the hyperoxaluria of the mankind is characterized in that daily is more than 40mg (about 440 μm of ol) or 30mg. Illustrative clinic cutoff level is, for example, male is 43mg/ days (about 475 μm of ol), women is 32mg/ days (about 350 μ mol).The urinary oxalate excretion that hyperoxaluria can also be defined as every gram of urinary creatine acid anhydride daily is more than 30mg.Slight hyperoxaluria Patient can drain at least 30-60 (342-684 μm of ol) or 40-60 (456-684 μm of ol) mg oxalates daily.Intestines originality top grass Uraturia patient can drain at least 80mg urinary oxalates (912 μm of ol) daily, and primary hyperoxaluria patient can drain daily At least 200mg (2280 μm of ol), for example, Borowski A.E, Langman CB.Hyperoxaluria and Oxalosis: Current Therapy and Future directions.Exp Opinion Phrama (2006, in printing).

The application of OXDC crystal and its composition

The application of oxalate decarboxylase according to the method for the present invention is not limited to any specific delivery system, including passing through upper stomach Enteron aisle such as mouth (such as in capsule, suspension, tablet or together with food) or the top of stomach or intestines (such as pass through conduit Or injection) administration, to reduce the levels of oxalate of individual.In some cases, endogenous oxalic acid brine is reduced using OXDC Flat and/or concentration.OXDC can also be provided by device outside, such as dialysis apparatus, the biological sample of conduit or contact from individual The structure or device of product.

Individual administration can be happened in single dose or repeat administration, and by it is a variety of it is physiologically acceptable in the form of in It is any and/or use the part (foregoing) of acceptable pharmaceutical carrier and/or additive as pharmaceutical composition.At this In the method for disclosure of the invention, oxalate decarboxylase can be individually, with one or more other bioactivators simultaneously or through weight Folded or nonoverlapping interval is continuously administered, and other bioactivators are for example, pydoxycin (vitamin B-6), orthophosphoric acid Salt, magnesium, glycosaminoglycan, calcium, iron, aluminium, magnesium, potassium citrate, Cholestyramine, organic marine products hydrocolloid, plant juice, for example, banana Stem juice or beet juice, or L-cysteine.Provide the biology of the activity or availability that reduce levels of oxalate or increase OXDC Activating agent.In successive administration, oxalate decarboxylase and one or more other reagents can be administered with any order.In some realities It applies in scheme, the length of section gap can be more than 2,4,6,12,24 or 48 weeks or more.

Oxalate decarboxylase can be used as unique reactive compound or with another reactive compound or combination of compositions Administration.Unless otherwise indicated, according to the progress oxalate decarboxylase of the seriousness of symptom and disease with about 10 μ g/kg to 25mg/kg Or the dosage administration of 100mg/kg.The suitable treatment effective dose of OXDC is selected by treating clinician, and approximate range is 10 μ g/kg to 20 mg/kg, 10 μ g/kg to 10mg/kg, 10 μ g/kg to 1mg/kg, 10 μ g/kg are to 100 μ g/kg, 100 μ g/kg To 1mg/kg, 100 μ g/kg to 10mg/kg, 500 μ g/kg to 5mg/kg, 500 μ g/kg to 20mg/kg, 1mg/kg to 5mg/kg, 1mg/kg to 25mg/kg, 5mg/kg are to 100mg/kg, and 5mg/kg to 50mg/kg, 5mg/kg to 25mg/kg and 10mg/kg are extremely 25mg/kg.Further, it is possible to use in embodiment or in Physician ' s Desk Reference (PDR) 2003,57th Ed., Medical Economics Company, the specific dosage pointed out in 2002.

The present invention oxalate decarboxylase crystal can for example, by oxalate decarboxylase is delivered to patient device outside or Catheter drug delivery.Conduit, for example, catheter, can be coated with the composition containing oxalate decarboxylase crystal.

The following examples provide the explanatory embodiment of the present invention.Those of ordinary skill in the art would appreciate that not The many improvement and variation that can be made in the case of changing the spirit or scope of the present invention.Such improvement and variation are included in In the scope of the present invention.Embodiment is not limit the invention in any way.

Embodiment

The fermentation and purifying of 1. oxalate decarboxylase of embodiment.

Oxalate decarboxylase (OXDC) from bacillus subtilis (B.subtilis) is by 6 identical monomer compositions Six glycoprotein polyprotein precursor of 261kDa homotypes.Each monomer contains 385 amino acid, and the molecular weight of calculating is~43-44kDa, isoelectric point It is 5.2.Using Bacillus subtilis genes group DNA as template, the OXDC genes of yvrK are referred to as before PCR amplification.

First by the OXDC gene clonings of amplification into pCRII carriers (Invitrogen, Carlsbad, CA), then sub- gram It is grand, and expressed using e. coli bl21 (DE3) pLysS cells from pET-11a expression vectors.It is this in pET-11a carriers Gene expression is under the control of T7 promoters, is induced by using IPTG (isopropyl-β-D-thiogalactoside), adjusts OXDC tables It reaches.

High level expressions of the OXDC of restructuring in Escherichia coli is realized using fermentation.Containing casein hydrolysate (USB Corporation, Cleveland, OH) or soy peptone, yeast extract (USB Corporation), NaCl (Fisher Scientific), PPG2000 antifoaming agent (PPG), KOH (Mallinckrodt Baker, Inc., Phillipsburg, NJ), and expressed in 800L (liter) fermentation medium of ampicillin (USB Corporation). Since OXDC is the enzyme of manganese-dependence, in the fermentation medium comprising 5mM MnCl₂·4H₂O(Mallinckrodt Baker, Inc.).By adding in 0.4mM IPTG (Lab Scientific), the expression of OXDC is induced.The cell growth of expression OXDC exists In shaking flask or fermentation tank.In this way, OXDC is mainly expressed in particulate preparation.

The Glycerol stock for the BL21 cells for using pET11a-OXDC conversions is used to ferment.It ferments, uses for 800L 2x250ml flasks prepare culture before seed, and each flask contains 50ml culture mediums (LB+100 μ g/ml ampicillins).It uses 2x0.5ml inoculum.35 DEG C, 250rpm culture culture 6 it is small when.Then the culture is transferred to 6x2L flasks, each Flask contains 1L culture mediums (LB+100 μ g/ml ampicillins).In each flask, 10ml starting cultures are added in.35 DEG C, 250rpm cultivate these cultures 12 it is small when.The 6L cultures are transferred to the fermentation tank that 800L contains above-mentioned appropriate culture medium In.Culture is grown in 37 DEG C, pH7.0, in 100rpm shakes dissolved oxygen is made to be about 30-40, until OD₆₀₀Reaching about 0.3 (should When process needs that about 2-3 is small).With 0.4mM IPTG+5mM MnCl₂·4H₂O induces the expression of OXDC.It is lured in 37 DEG C, 100rpm Lead culture 4 it is small when.Then cell is harvested, and is freezed spare.OD in harvest₆₀₀It is about 5.5-8.0.

In the presence of 15-25U/mlDNA enzymes are with or without, with 1kg cells paste/4L contain 50mM Tris pH8, The ratio of the buffer solution of 100mMNaCl, again suspension cell.It is overnight that (50-60rpm) cell suspending liquid is mixed at 4 DEG C.Make cell Suspension passes through the homogenizer 3 times in precooling on ice.Cell cracking efficiency is checked under the microscope, by unbroken cell Suspension is used as control.At 4 DEG C, in 1L bottles, in 4,000rpm centrifuge cells 40min.Supernatant and particle are preserved, is used for SDS-PAGE is characterized.By SDS-PAGE, the expression of OXDC is analyzed.Most OXDC is found in the grain, including forgiving Body and other precipitations.By centrifuging 40min in 4000rpm, particle is harvested, it is used immediately or spare in -70 DEG C of freezings.

From 800L fermentation reactions, we obtain 5,000 to 5,500g cells, they generate 2,800 to 3,000g weight in wet bases Grain.

Embodiment 2. is with mild denaturant concentration and subsequent anion-exchange chromatography from the grain crystalline oxalic acid of dissolvingization Decarboxylase.

OXDC crystal will be used to prepare in the OXDC particles of -20 DEG C of refrigerations.

In this operation, the dissolved particles under the denaturant concentration and pH of temperate condition.Then anion exchange base is used Matter column folds the albumen of dissolvingization again.

By grain dissolution in 2M ureas, 100mM Tris pH10.0,10mM DTT and 100mM NaCl (1:10w/v).In room Temperature (RT) agitating solution 2h, then at 4 DEG C in 15K centrifugation solution 30min.Supernatant, and preservation is carefully decanted.Carefully will Particle is weighed, preservation respectively.

Under constant and gentle agitation, with the flow velocity of 10ml/min, the particle of the dissolvingization in supernatant is added dropwise 10 volumes by 2M ureas, 100mM Tris pH8.0,1mM DTT and 1mM MnCl₂In the solution of composition.Supernatant is added in terminate Afterwards, in incubation at room temperature protein solution 1h.After incubation, by centrifuging 30min at 4 DEG C, in 15K, to remove arbitrary possible precipitation, Protein solution is got out for anion-exchange chromatography.

By the way that Q agarose matrix is packed in glass column, anion-exchange column is prepared.The column is connected to FPLC On, by using the 0.5M ureas of 10 column volumes (CV), 100mM Tris pH8.0,1mM DTT and 1mM MnCl₂Solution washs, into Row balance.Flow velocity maintains 6ml/min.The particle of dissolvingization is loaded into upper prop.Protein load is 8-10mg/ml matrix.It loads After sample, in the flow velocity of 6ml/min, with 100mM Tris pH8.0, the 1mM DTT of at least 10 column volumes and 1mM MnCl₂It washes Wash column.The step removes urea from the protein sample of combination, and albumen is made to be folded into its native conformation again.

By discontinuous gradient or stepwise elution, eluted with 1M NaCl, 100mM Tris pH8.0 and 1mM DTT from column Albumen.Albumen wash-out is monitored in 280nm, collects 10ml fractions.Peak fraction is merged together, passes through SDS-PAGE and enzymatic activity It is tested.

The peak fraction containing OXDC is concentrated into 15mg/ml as follows：Cell is stirred using 10,000MWCO films, in 10 volumes 100mM Tris pH8.0,100mM NaCl and 1mM DTT in dialyse.With 1 it is small when interval, change buffer solution 2 times, third time After changing buffer solution, continue dialysed overnight, to realize the maximum crystal rate of recovery.By at 4 DEG C, in 15 min of 2K Centrifuge A samples, Recycle the albumen crystallized in bag filter.After dialysis, the OXDC of about 70% refolding can be crystallized.Crystal is cubic shaped, tool There is homogeneous size.Crystal shows the activity of about 44 units.

Embodiment 3. uses denatured agent concentration and subsequent anion-exchange chromatography, makes oxalic acid decarboxylation by dissolved particles Enzyme crystallizes.

In this way, particle is dissolved in 5M ureas, 50mM Tris pH8.6,100mM NaCl, 10mM DTT (1:5w/ v).Solution 2h is stirred at room temperature, is then centrifuging solution 30min at 4 DEG C, in 15K.Supernatant, and preservation is carefully decanted.It is small Heart weighs particle, preservation respectively.

By the way that Q agarose matrix is packed in glass column, anion-exchange column is prepared.The column is connected to FPLC On, it washs, is balanced by using the 4M ureas of 3 column volumes (CV), 100mM Tris pH8.6 and 10mM DTT.With 7 column volumes 100mM NaCl, 50mM Tris pH8,1mM MnCl₂, 10mM DTT, further column scrubber, in a single step with 3 columns 0.5MNaCl, 50mMTrispH8.0,1mMDTT, 1mMMnCl of volume₂Elution.

Appropriate fraction is collected, albumen is identified by SDS-PAGE and determination of activity.It is deposited there is (0.5MNaCl) with high salt Under, soluble albumen is concentrated.With 0.5M eluting salt 450ml total volumes, through pellicon filtering and concentratings to 45ml, enter 100mMNaCl、50mMTrispH8.0、1mMDTT.It after protein concentration, is diluted, so that salinity is made to be reduced to from 0.5M 0.1MNaCl.At this moment, OXDC crystal initially forms.In this embodiment, 100mMNaCl, 50mMTrispH8.0, In 1mMDTT equivalents, volume is adjusted to 210mL.The crystal formed is rotated, in the presence of 1mMDTT is with or without, It is recycled in 100mMNaCl and 50mMTrispH8.0.

Embodiment 4. is made using high pH and mild denaturant concentration, then progress doughnut concentration by dissolved particles Oxalate decarboxylase crystallizes.

In room temperature, by the particle (3.93kg) containing inclusion body and other precipitations be dissolved in 9.5L50mM Tris pH12, When 500mMNaCl, 2M urea, 10mMDTT2 are small.Final volume is 12L, pH9.9.Sample is rotated in 7000rpm 45 minutes, in recycling Clear liquid.Postrotational total volume is 11.1L, pH9.9.On the hollow fibers by sample concentration to 5L1 it is small when, Ran Houyong Volume is slowly adjusted to 20L by 50mMTrispH8.0,500mMNaCl, 2M urea, 10mMDTT.When process progress 1 is small.Herein When, the final concentration for estimating urea is about 0.5M ureas.By volume concentration to 6L2.5h in doughnut.With 50mMTrispH8.0, 500mMNaCl、1mMDTT、1mMMnCl₂, 200mML- arginine, sample is diluted to 24L30min.At this moment, urea is estimated Concentration is 125mM.Another wheel concentration is carried out, until final volume 5L.With 50mMTrispH8.0,500mMNaCl, 1mMDTT, 1mMMnCl₂, sample is diluted to 18L, is again concentrated to 6.5L.PH is 8.1 at this time.Sample is rotated in 7000rpm 45 minutes, Final particle is preserved for analyzing.After centrifugation, step is diluted with 50mMTrispH8.0,1mMDTT, obtains crystal. Room temperature under mixing, 30L is diluted to using peristaltic pump.The flow velocity for estimating dilution is 50ml/min.It is small that the dilution carries out about 9 When.By the way that crystal is collected by centrifugation, preservation supernatant is used to analyze.Crystal is washed with 50mMTris, 100,mMN,aCl,pH8 3 times, weight Newly it is suspended in 50mMTris, 100mMNaClpH8.In 4 DEG C of preservation crystal.

Embodiment 5. makes oxalate decarboxylase be crystallized from cell extract using with high salt

(1)Using particle of the high salt concentration dissolving containing albumen, then concentration and dilution, crystallized

In room temperature, by the frozen particle (465g) of embodiment 1 be dissolved in 2.3L100mMTris, 1mML- cysteine HCL, In 0.5MNaCl, pH8.0 2 it is small when, form soluble oxalate decarboxylase.Sample is rotated in 7000rpm 45 minutes, recycles supernatant Liquid.Final volume is 2.15L, and the protein concentration measured is 24.14mg/ml.It is by grossflow filtration (10kDPall), sample is dense Be reduced to 550ml1 it is small when, then in the flow velocity of 73ml/min, through 30 minutes with 100mMTrispH8.0,1mML- cysteine HCl Be diluted to 2,750L, be stirred at room temperature 1 it is small when.Form crystal overnight in cold house.By the way that crystal, preservation supernatant is harvested by centrifugation For analyzing.Wash crystal 3 times with 100mMTris, 100mMNaClpH8, be then resuspended in 100mMTris, 100mMNaClpH8.In 4 DEG C of preservation crystal.The recombined bacillus subtilis OXDC purified from Bacillus coli expression culture medium is being marked The ratio that about 50-60U/mg is shown under quasi- determination condition (referring to embodiment 15) is lived.

(2)Using high salt concentration dissolved particles, then concentration and dialysis, crystallized

In room temperature, by the frozen particle (510g) of embodiment 1 be dissolved in 3L100mMTris, 1mML- cysteine HCL, In 0.5MNaCl, pH8.0 2 it is small when.Sample is rotated in 7000rpm 30 minutes, recycles supernatant.Pass through grossflow filtration (10kDPall), by sample concentration to 500ml1 it is small when, then dialyse under stiring in 100mMTrispH8.0.In cold house Crystal is formed overnight.By the way that crystal is harvested by centrifugation, preservation supernatant is used to analyze.Crystal 3 is washed with 100mMTris, pH8.0 It is secondary, then it is resuspended in 100mMTrispH8.0.In 4 DEG C of preservation crystal.Crystal yield is 60%.

(3)Using high salt concentration dissolved particles, then concentration and dialysis, crystallized

In room temperature, by the frozen particle (510g) of embodiment 1 be dissolved in 3L100mMTris, 1mML- cysteine HCL, In 0.5MNaCl, pH8.0 2 it is small when.Sample is rotated in 7000rpm 30 minutes, recycles supernatant.Pass through grossflow filtration (10kDPall), by sample concentration to 500ml1 it is small when, then dialyse under stiring in 100mM Tris pH7.5.In cold house In form crystal overnight.By the way that crystal is harvested by centrifugation, preservation supernatant is used to analyze.Crystal is washed with 100mM Tris, pH7.5 3 times, then it is resuspended in 100mM Tris pH7.5.In 4 DEG C of preservation crystal.Crystal yield is 67%.

(4)Using high salt concentration dissolved particles, then concentration and dialysis, crystallized

In room temperature, by the frozen particle (510g) of embodiment 1 be dissolved in 3L100mM Tris, 1mM L-cysteines HCL, In 0.5MNaCl, pH8.0 2 it is small when.Sample is rotated in 7000rpm 30 minutes, recycles supernatant.Pass through grossflow filtration (10kDPall), by sample concentration to 500ml1 it is small when, then dialyse under stiring in 100mM Tris pH7.0.In cold house In form crystal overnight.By the way that crystal is harvested by centrifugation, preservation supernatant is used to analyze.Crystalline substance is washed with 100 mM Tris, pH7.0 Then body 3 times is resuspended in 100mM Tris pH7.0.In 4 DEG C of preservation crystal.Crystal yield is about 80%.

(5)Using high salt concentration dissolved particles, then concentration and dialysis, crystallized

In room temperature, by the frozen particle (510g) of embodiment 1 be dissolved in 3L100mM Tris, 1mM L-cysteines HCL, In 0.5M NaCl, pH8.0 2 it is small when.Sample is rotated in 7000rpm 30 minutes, recycles supernatant.Pass through grossflow filtration (10kD Pall), by sample concentration to 500ml1 it is small when, then dialyse under stiring in 100mM sodium citrate buffer solutions pH6.5. Crystal is formed in cold house overnight.By the way that crystal is harvested by centrifugation, preservation supernatant is used to analyze.With 100mM sodium citrate buffer solutions PH6.5 washings crystal 3 times, is then resuspended in 100mM sodium citrate buffer solutions pH6.5.In 4 DEG C of preservation crystal.Crystal produces Rate is about 70%.

(6)Using high salt concentration dissolved particles, then concentration and dialysis, crystallized

In room temperature, by the frozen particle (510g) of embodiment 1 be dissolved in 3L100mM Tris, 1mM L-cysteines HCL, In 0.5MNaCl, pH8.0 2 it is small when.Sample is rotated in 7000rpm 30 minutes, recycles supernatant.Pass through grossflow filtration (10kDPall), by sample concentration to 500ml1 it is small when, then under stiring in 100mM sodium citrate buffer solutions pH6.0 thoroughly Analysis.Form crystal overnight in cold house.By the way that crystal is harvested by centrifugation, preservation supernatant is used to analyze.Delayed with 100mM sodium citrates Fliud flushing pH6.0 washings crystal 3 times, is then resuspended in 100mM sodium citrate buffer solutions pH6.0.In 4 DEG C of preservation crystal.It is brilliant Body yield is about 60%.

(7)After homogenization and dissolving, the OXDC from cell paste is crystallized

With the ratio of 1kg cells paste/3L buffer solutions again suspension cell, the buffer solution contains 100mM Tris PH7.5,500mM NaCl, 5mM cysteines and 1mM manganese chlorides.It is overnight that (50-60rpm) cell suspending liquid is mixed at 4 DEG C.Make Cell suspending liquid passes through the homogenizer 2 times in precooling on ice.Cell cracking efficiency is checked under the microscope, it will be unbroken thin Born of the same parents' suspension is used as control.Suspension is complemented into 10L, when room temperature is small with outstanding top blender mixing 3.Then at 4 DEG C, 7, 000rpm centrifuges crude extract 30min, recycles supernatant.Supernatant and particle are preserved, is characterized for SDS-PAGE.Pass through SDS- PAGE analyzes the expression of OXDC.Most OXDC is found in supernatant.Final volume is 10L, and the protein concentration of measurement is 34mg/ml.By grossflow filtration (10kD Pall), by sample concentration to 3.5L1 it is small when, then make under stiring in room temperature With crystallization buffer (100mM Tris, 100mM NaClpH7.5) dilution 1 it is small when.By the way that crystal, preservation supernatant is harvested by centrifugation For analyzing.Wash crystal 3 times with 100 mM Tris, 100mM NaClpH7.5, be then resuspended in 100mM Tris and 100mM NaCl、pH7.5.In 4 DEG C of preservation crystal.

Embodiment 6. crystallizes the OXDC from soluble protein。

In shaking flask, in expression in escherichia coli OXDC.Using 25mMTris-HCl pH of buffer 8.0, contain 25U/ The 100mMNaCl of mlDNA enzymes I, with Micro Fluid instrument（microfluidizer）Cell lysis.In incubation at room temperature cell lysate 1 Hour, make OXDC Crystallizations.Rotating crystal is rebuild in 100mM Tris, 100mM NaClpH8.

Embodiment 7. passes through Vapor diffusion crystallization OXDC.

Use commercially available sparse matrix crystalline reagents box:Crystal Screen(Hampton Research; Aliso Viejo,CA),Crystal Screen 2(Hampton Research),Wizard I(Emerald Biosystems;Bainbridge Island,WA), WizardII(Emerald Biosystems),CryoI(Emerald Biosystems) and CryoII (Emerald Biosystems), hanging-drop crystallization trials are carried out.

600 μ l reagents are placed in each hole.3 μ l reagents are dripped on microscope glass coverslip, 3 μ lOXDC are dripped to In reagent droplet, it is gently mixed.From the 6 μ l reagents and OXDC drops, other 5 drops are prepared.With being gently mixed for drop, Each subsequent (smaller) drop has different and unknown albumen/reagent ratio, is obtained in a short period of time so as to increase The possibility of crystal.After incubation at room temperature is stayed overnight, the crystal of hanging drop is checked under the microscope.Mass crystallization condition is obtained, such as table 1 It is shown.

Table 1:The crystallization condition of OXDC in hanging drop^a

^aIt is about 1.7mg/mL to measure OXDC concentration by Bradford experiments.

Embodiment 8. passes through micro- batch（microbatch）Crystallize OXDC.

By micro- batch of method, oxalate decarboxylase is crystallized from many crystallization conditions:

(i) mix the OXDC and 10 μ l16%PEG8000 that the 10 μ l that concentration is 23.46mg/ml are purified.It is stood in 2-5 seconds Crystallize.The big crystal formed contains some precipitations.

(ii) mix the OXDC and 10 μ l20%PEG8000 that the 10 μ l that concentration is 23.46mg/ml are purified.In 2-5 seconds It crystallizes immediately.The big crystal formed does not contain precipitation.

(iii) mix the OXDC and 10 μ l24%PEG8000 that the 10 μ l that concentration is 23.46mg/ml are purified.In 2-5 seconds It crystallizes immediately.Form the crystal of smaller cubic shaped.

(iv) mix the OXDC and 10 μ l28%PEG8000 that the 10 μ l that concentration is 23.46mg/ml are purified.In 2-5 seconds It crystallizes immediately.Form the crystal of very small cubic shaped.Do not precipitate.

(v) mix the OXDC and 12 μ l24%PEG8000 that the 8 μ l that concentration is 23.46mg/ml are purified.It is stood in 2-5 seconds Crystallize.Form the crystal of very small cubic shaped.Do not precipitate.

(vi) mix the OXDC and 11 μ l24%PEG8000 that the 9 μ l that concentration is 23.46mg/ml are purified.It is stood in 2-5 seconds Crystallize.Form the crystal of small cubes shape.Do not precipitate.

(vii) oxalate decarboxylase that the 10 μ l that concentration is 23.46mg/ml are purified is made to be mixed with 10 μ l24%PEG8000. It is crystallized immediately in 2-5 seconds.Form the crystal of small cubes shape.Do not precipitate.

(viii) oxalate decarboxylase that the 11 μ l that concentration is 23.46mg/ml are purified is made to be mixed with 9 μ l24%PEG8000. It is crystallized immediately in 2-5 seconds.Form the crystal of cubic shaped.Do not precipitate.

(ix) mix the OXDC and 8 μ l24%PEG8000 that the 12 μ l that concentration is 23.46mg/ml are purified.It is stood in 2-5 seconds Crystallize.Form the crystal of cubic shaped.Do not precipitate.

The activity of 9. soluble OXDC and OXDC crystal of embodiment

After dissolved particles, soluble OXDC is collected as described in Example 5, is rotated, and is recycled supernatant.Harvest and washing are brilliant After body, OXDC crystal is collected as described in Example 5.According to embodiment 15, the activity of soluble OXDC and OXDC crystal is measured. In one experiment, the activity of soluble OXDC is 12 units/mg, and the activity of OXDC crystal is 35 units/mg.

Soluble OXDC, and the harvest as described in any of embodiment 2-8 are collected as described in any of embodiment 2-5 OXDC crystal.According to embodiment 15, the soluble activity with crystal OXDC of measurement.The activity of OXDC crystal can be soluble At least about 100%, 200%, 300%, 400% or 500% of the activity of OXDC.

Embodiment 10. uses glutaraldehyde cross-linking oxalate decarboxylase crystal.

The oxalate decarboxylase crystal prepared using glutaraldehyde cross-linking according to any of embodiment 2-8.It, will after crystallization OXDC crystal is concentrated into 20-30mg/ml.0.8ml25% glutaraldehydes are added in into 20ml crystal, 1% glutaraldehyde solution are made, in room temperature When the crystal 18 that rolls is small.Crosslinked crystal is washed with 100mM Tris, pH7.00 5 times, is resuspended in 10mMTris, pH7.00 In.

The ratio for comparing lenticular OXDC and crosslinked OXDC (being referred to as OXDC-CLEC) is lived (6 experiments), is shown different In product, original activity that crosslinked oxalate decarboxylase crystal retains lenticular albumen is more than 30% to more than 50%.

In order to test influence of the glutaraldehyde to enzymatic activity of various concentration, in pH8.0, at 25 DEG C, with the penta of various concentration When the 1ml aliquots (60mg/ml) 18 of dialdehyde (from 0.05% to 2%, final concentration) crosslinking OXDC crystal are small.By in Ai Pengdao Crosslinked crystal is separated in husband's pipe in 2000rpm centrifugations, so as to terminate crosslinking, is then resuspended in crosslinked crystal In 100mM TrisHCl, the pH7.0 of 1ml.Then crosslinked OXDC is washed with 100mM TrisHCl pH of buffer 7.5 (OXDC-CLEC) 5 times, 3 times (referring to the results of table 2 below) then are washed with 10mM TrisHCl pH of buffer 7.5.

The solubility of the pH controls of 11. crosslinked oxalate decarboxylase crystal of embodiment.

PH is made to be reduced to 3.0 from 7.5, checks the solubility of different crosslinked oxalate decarboxylase crystal.In 1mg/ml, Crosslinked crystal is incubated in 50mM glycine HCl (pH3.0).Under stiring, after when 37 DEG C of incubations 5 are small, taking-up etc. point examination Sample.After 2000rpm centrifuges not molten crosslinking crystals and passes through 0.22 μm of filter filtering supernatant, in OD₂₈₀Nm is measured Soluble protein concentration.As a result as described in following table 2.

Table 2:The solubility controlled with the glutaraldehyde cross-linking OXDC crystal of different weight percentage and the pH of OXDC-CLEC

Sample	% glutaraldehydes	% albumen leaches
			OXDc-CLEC-1	0.005	100.0
OXDC-CLEC-2	0.010	100.0
			OXDC-CLEC-3	0.050	2.2
OXDC-CLEC-4	0.075	0.0
			OXDC-CLEC-5	0.100	0.0
OXDC-CLEC-6	0.200	0.0
			OXDC-CLEC-7	1.000	0.0

These results indicate that in the presence of at least about 0.05% (final concentration) glutaraldehyde, penta substantially stablized is formed The crosslinked crystal of dialdehyde OXDC.

12. soluble OXDC of embodiment, the pH living features of lenticular OXDC and OXDC-CLEC

By adding in glutaraldehyde (Sigma), the oxalate decarboxylase crystal prepared as described in embodiment 2-8 is crosslinked.At 25 DEG C, With the 1ml aliquots (30-40mg/ml) 18 of 1% glutaraldehyde (final concentration) of pH8.0 crosslinking OXDC crystal it is small when.By angstrom Peng doffer Guan Zhong 2000rpm centrifuge to separate crosslinked crystal, and so as to terminate crosslinking, then crosslinked crystal suspends again In 1ml100mMTrisHCl, pH7.0.Then washed OXDC-CLEC5 times with 100mMTrisHCl pH of buffer 7.0, with It is washed 3 times with 10mM TrisHCl pH of buffer 7.0 afterwards.Using different buffer solution and pH, by as described in Example 15 The activity of crystal is measured, measures the pH living features of OXDC-CLEC:50mM glycine/HCl buffers, in pH2.0 and 3.0; 50mM Succinate Buffers, in pH4.0,5.0 and 6.0;With 50mM Tris buffer solutions, in pH 7.0.It measures in each pH Activity level 2 times, calculate average activity.It is shown in FIG. 1 the result shows that, OXDC-CLEC between pH3.5 to 6.0 than it The active higher of solvable counterpart.Uncrosslinked crystal shows more much higher than the soluble form of oxalate decarboxylase in different pH Activity, from the higher of about 50% to about 200%, 300% or 400%.

Oxalate decarboxylase treatment in 13. intestines originality hyperoxaluria animal model of embodiment.

The rat model of intestines originality hyperoxaluria, dosage range research:Feed the male Sprague of high oxalates diet Dawley (SD) rat forms the animal system for being suitable for research intestines originality hyperoxaluria.In this study, using 1.1% diet Potassium oxalate causes urinary oxalate to increase by 5 to 10 times.

It is less than 35 day age by 20 and weight is randomly divided into control for 100-120 grams Sprague Dawley (SD) rat Group and experimental group (every group of 5 rats).Before treatment, rat is made to adapt to respective metabolic cage (LabProducts, Inc.; Seaford, DE) 7 days.At this stage, unlimitedly supply the acidifying water of supplement to rat, and feed containing 1.1% potassium oxalate and Synthesis diet (the Research Diets TD89222PWD of low (0.5%) concentration calcium;Harlan Teklad;Madison,WI). During treatment, rat maintains the diet.

After laundering period, 3 various doses of crosslinking crystals (see, e.g., embodiment 9) will be configured to 1% glutaraldehyde Restructuring oxalate decarboxylase be administered to the continuous week of experimental rat 4.Crystal as solidification/dry food enzymatic mixture mouth Clothes (5,25 and 80mgOXDC-CLEC slurries mix in 10mM TrisHClpH7.0 with 15g foods independently, and Sets/dries;Each morning recharges about 20g foods/enzymatic mixture to food container).Before treatment, based on them Basic urinary oxalate, rat is randomly divided into control group and experimental group.

The analysis of urine sample:In the metabolic cage of acid (250 μ l6N hydrochloric acid is made to be mixed with the urine sample in middle collection for 24 hours) above Twenty-four-hour urine sample is collected, so that urine ascorbic acid is made to be minimized to the Auto-decomposition of oxalates.Before further analysis ,- 70 DEG C of preservation samples.Daily urine and multiple (hour) urine samples for 24 hours are collected, is measured for oxalates and kreatinin.Oxalates and The measure of kreatinin is as described in Example 15.The homaluria of oxalates and kreatinin is expressed as what is detected in urine sample for 24 hours μm ol of oxalates and kreatinin.It is examined using Studentt-, all data of statistical analysis.

As shown in Fig. 2, to the SD Oral Administration in Rats OXDC-CLEC of chronic hyperoxaluria, cause to urinate grass since treating the 4th day The lasting reduction of hydrochlorate.The continuous reduction of maximum of 25-40% is recorded in maximum dose level group (80mg OXDC-CLEC).Lower dose 5mg the and 25mg OXDC-CLEC of amount cause the smaller of urinary oxalate reduce (in 25mg groups be up to 30%, 5mg groups in up to 20%).The dosage of 25mg and 80mg is generated in all test days (except 25mg groups the 21st beyond the highest heavens) and significantly reduced, while most Low dosage 5mg has inapparent smallest effect.The result discloses the dose dependent effect of OXDC-CLEC treatments.

Embodiment 14:Oral oxalate decarboxylase treatment in primary hyperoxaluria animal model.

The mouse model of I type primary hyperoxalurias:The mouse that AGT1 is knocked out lacks liver peroxidase alanine:Acetaldehyde Sour aminopherase, that is, the defects of causing I type primary hyperoxalurias.By gomphosis mouse in C57Bl6 and 129/sv background strains It is multiplied into homozygous line.Compared with wild type (0.2mmol/L), the Agxt mouse of all homozygosis show slight hyperoxaluria (1-2mmol/L), urinary oxalate raise 5-10 times than normal value., it was also found that the male of 30-50% and 0% female are after life Phase (4-7 monthly ages) develops into the calcinm oxalate calculus of slight nephrocalcinosis and urethra.Interestingly, when of the same race When mutation is analyzed in C57Bl6 plants, the hyperoxaluria in any one gender is uncorrelated to urinary calculus development；This address in the disease In the phenotypic variability that is frequently observed.

In these experiments using (strain AGT1 KO/129sv, developer are Dr.Salido, La to totally 44 male mices Laguna Tenerife,Spain).Mouse is randomly divided into control group and 3 experimental groups.Mice weights are 20-25 grams, are less than 6 monthly ages.

Spent glycol (EG) attacks AGT1 KO (129sv) mouse, to cause serious hyperoxaluria and the shape in kidney essence Into calcium oxalate deposits object.EG is a kind of common alcohol that oxalates is metabolized in liver.In general, after EG is attacked 2-6 weeks, 129/ AGT1 KO mouse in sv backgrounds show impaired renal function sign, this can be measured as follows：(i) change of Oxalate is urinated The excretion of change；(ii) creatinine clearance and (iii) reduced ultimately results in kidney failure and dead nephrocalcinosis.

Before treatment, mouse is made to adapt to each metabolic cage (Tecniplast USA Inc, Exton, PA, USA) 7 days, Feeding containing be less than 0.02-0.08% oxalates and about 0.5-0.9% calcium standard breeding stock diet (17% albumen, 11% is fatty, 53.5% carbohydrate).After laundering period, mouse is divided into 4 groups;The oxalic acid decarboxylation that 3 treatment group's feedings are mixed with food Enzyme-CLEC, and the mouse in matched control group receives the identical diet without adding in experiment object.From treatment first day to grinding Study carefully end, the drinking water for being with the addition of 0.7%EG is unlimitedly provided to all mouse.Attack after a few days, mouse daily they About 3-6mmol/L oxalates is drained in urine, it is about 10-20 times high that this than wild type (does not attack) mouse.

The application of OXDC-CLEC enzymes;Dosage range is studied:In the dose study of OXDC-CLEC, it only is from using totally 44 AGT1KO/129sv plants of male mice.Mice weights are 20-25 grams, less than 6 monthly ages.Mouse is attacked with EG, then random point Into control group and experimental group.Monitoring is configured to crosslinking crystals (1% glutaraldehyde;Referring to embodiment 9) 3 various doses restructuring The effect of oxalate decarboxylase 4 in continuous week.The term " OXDC-CLEC " used in this embodiment refers to as described in Example 9 It is configured to the restructuring oxalate decarboxylase of crosslinking crystals (1% glutaraldehyde).In 5,25 and 80 nominal standard doses of mg/ days, OXDC-CLEC As solidification/dry food enzymatic mixture takes orally.By sufficient amount in 10mM Tris-HCl buffer solutions (pH7.0) Enzyme slurry is mixed with 3.5g foods, and sets/dries.Each morning recharges that about 7g foods/enzyme mixes to food container Object.

The assessment of the effect of OXDC-CLEC:Reduced by urinary oxalate, the prevention of deposition of the calcium oxalate in kidney essence and The effect of survival rate, monitoring enzyme treatment.At the end of the study, the mouse of survival is put to death, blood sampling measures for kreatinin.

The analysis of urine sample：Twenty-four-hour urine sample is collected in the metabolic cage of acid (50 μ l6N hydrochloric acid/3-4ml urine) above, so as to Urine ascorbic acid is made to be minimized to the Auto-decomposition of oxalates.Before further analysis, in -20 DEG C of preservation urine samples.It collects every It urine and multiple urine samples for 24 hours analyze oxalates and creatinine levels.The measure of oxalates and kreatinin such as 15 institute of embodiment It states.The homaluria of oxalates and kreatinin is expressed as being excreted to μm ol of the oxalates and kreatinin in urine sample (mL) for 24 hours.Make It is examined with Studentt-, statistical data analysis.

The analysis of blood sample:At the end of the study, mouse is put to death, collects blood serum sample.It measures, uses for serum creatinine The Jaffe reaction methods of slight improvement are (see, e.g. the creatine from Oxford Medical Research, Inc. Acid anhydride microdetermination tablet kit;Slot,Scand J.Clin.Lab.Invest.17:381,1965;And HeinegardD, Clin.Chim.Acta43:305,1973).The 80 undiluted blood serum samples of μ l and 800 μ l picrins are mixed in test tube, In incubation at room temperature 30 minutes.It measures and develops the color in 510nm spectrophotometer methods；Then 33.3 μ l60% acetic acid are added in, non-spy is quenched Opposite sex reaction.Sample is thoroughly mixed, again in 510nm readings in incubation at room temperature after five minutes.Final absorbance is expressed as 2 The difference of secondary reading.The serial dilutions of 1mM creatine anhydride solutions are used for standard curve.

By measuring creatinine clearance, renal function is measured indirectly.Creatinine clearance is expressed as to the excretion of kreatinin Rate (U_crxV)（Wherein U_crRepresent the Concentrations (μm ol/L) in urine sample）Divided by plasma creatinine (P_cr).This is expressed as:

C_cr=(U_crxV)/P_cr=mL/h

The security parameter monitored in the course of the research is the death rate, food and water intake and weight.In the course of the research, The objective observation of death rate inspection and cage side is carried out daily once.Measurement food intake daily records weekly water intake.It is opened in research During the beginning and at the end of research, the weight of all animals is recorded.

As shown in figure 3, to EG attack AGT1KO (129sv) Mouse oral OXDC-CLEC, cause from treatment the 4th day to At the end of research, compared with matched untreated control mice, urinary oxalate is horizontal to be significantly reduced.In all 3 treatments 30 to 50% reduction is observed in group, maximum reduction is observed in maximum dose level group (80mgOXDC-CLEC).25mg and The urinary oxalate that the lower dosage of 5mgOXDC-CLEC generates up to 35% reduces.

It is examined by azygous double tail Studentt-, analysis result.Study start when, each administration group have n= 11 mouse, but several mouse die of ethylene glycol attack in the course of the research.Shown result is only included in urinary oxalate measurement Certain day survival mouse.Observe urinary oxalate starting rise cause nephrocalcinosis, reduce kidney filtering function and Final dead under the reduction at any time of subsequent oxalates discharge rate and worst condition, they can best explain control The bell shaped curves that urinary oxalate is drained in group.

Assessment renal function is measured by creatinine clearance.At the end of the study, put to death live through EG attack 4 weeks it is all move Object collects blood to measure plasma creatinine and creatinine clearance.All 11 mouse of 80mg dosage groups live through EG attacks 4 Week;8/11 mouse survival of 25mg OXCD-CLEC dosage groups;8/11 mouse survival of 5mgOXCD-CLEC dosage groups；It is right According to 7/11 mouse survival of group.It is measured for serum creatinine, uses the Jaffe reaction methods of above-mentioned slight improvement (see, e.g. the kreatinin microdetermination tablet kit from Oxford Medical Research, Inc.;Slot, Scand J.Clin.Lab.Invest.17:381,1965;With Heinegard D, Clin.Chim.Acta 43:305, 1973)。

By measuring creatinine clearance, the effect of oral OXDC-CLEC is to renal function is assessed.Live through entire moon research The creatinine clearance of the mouse in stage is as shown in Figure 4.When being compared with control group, the survival for receiving 80mg OXDC-CLEC is small Notable higher (the p of creatinine clearance of mouse<0.05).

All mouse (11/11) of 80mg OXDC-CLEC treatment groups live through 4 weeks EG attack options, and only 7 in control group Mouse (7/11) lives through the program.The kidney filtering rate measured by creatinine clearance is also significantly lower than 80mg dosage groups (Fig. 4).

Renal pathology credit is analysed:Paraffin embedding routinely handles Mouse Kidney, and positions, to obtain the complete crosscutting of kidney Piece.With each 4 μm of section, each kidney is cut into 12 serial section, is dyed with hematoxylin and eosin, is examined for routine histologic It looks into or presence of the calcium oxalate crystals in nephridial tissue is detected by specific Yasue metal replacements histochemical method.It uses 20X magnifying powers, check glass slide under the microscope, and inspection personnel scores section under 4- class scales, and identical standard is applied to Each anatomic region (cortex, medullary substance and mastoid process) of kidney.Scoring is (i) without (not having oxalic acid salt crystal in any region);(ii) At least (there is 1-5 crystal in arbitrary region);(iii) medium (having 6-10 crystal in arbitrary region);(iv) is serious (all Region has multiple crystal set).

The presentation graphics of nephridial tissue from treatment and control-animal is as shown in figures 5a-5c.In 20x magnifying powers, in kidney The calcium oxalate crystals of the visible Yasue- positives in essence.Have with all mouse for the treatment of group of 80mg OXDC-CLEC treatments normal Healthy kidney, without calcium oxalate deposits object trace (Fig. 5 A).In control group and in some mouse from low dose therapy group In, observe medium nephrocalcinosis (Fig. 5 B) and serious nephrocalcinosis (Fig. 5 C).White arrow instruction oxalic acid in Fig. 5 C Doped calcium object, big region of the grey arrow instruction with interstitial fibrosis.

The histological examination of kidney is disclosed using specific Yasue metal replacements method, deposit is primarily present in kidney Cortex and medullary substance part.In the case of serious nephrocalcinosis (Fig. 5 C), calcium oxalate deposits object random distribution in kidney.Also see The sign of fibrosis and inflammation, the morphologic change of glomerulus are observed, calcium oxalate deposits object is formed once in a while in glomerulus.In corpse After dissect, all mouse (11/11) from 80mg dosage groups have normal kidney form, without calcium oxalate in kidney or bladder The trace of deposition.On the contrary, the mouse of 100% (11/11) from untreated control group has calcium oxalate deposits object.It is controlled low Treatment group (25mg or 5mgOXDC-CLEC), the mouse of 63% (7/11) have calcium oxalate deposits object in kidney.These results confirm OXDC-CLEC oral medications are to the reduction of hyperoxaluria and kidney calcium oxalate crystal growth in healthy crystal deposition in the AGT1 KO mouse of EG attacks Positive, the dose-dependent effect of prevention.The summary of histopathological analysis from all 4 groups of mouse is as shown in table 3.

It is involved after the seriousness of 3. nephrocalcinosis of table and OXDC-CLEC oral medications in treatment group and control group mouse Number

Also OXDC-CLEC oral medications are had rated to control mice and the urinary calculus of the mouse from 3 different treatment groups The effect of forming frequency.2 major class calcium are found that in the bladder from EGAGT1KO mouse:Calcium oxalate monohydrate calculus and grass Sour calcium dihydrate calculus.In 36% (4/11) mouse of control group and in 19% (2/11) mouse of 2 Ge Gengdi treatment groups, In the presence of being evident that vesical calculus.Vesical calculus is not observed in 80mg high dose OXDC-CLEC groups.X-ray diffraction Analysis shows, the calculus with albescent, coarse bud shape surface are mainly made of calcium oxalate monohydrate, and with sharp keen crystalline substance The calculus of the opposite bigger at body angle corresponds to oxalic acid calcium dihydrate.

Survival rate analysis is carried out by Kaplan-Meier estimators.Using Kaplan-Meier methods, wherein will be at certain A little survival rates of subject of time point death divided by the number for the subject still survived at the moment under study for action, analysis Influence of the OXDC-CLEC treatments to the survival rate of the mouse of ethylene glycol attack.In the research of this method graphic rendition between group Difference (Fig. 6).Statistics program such as Kaleida schemes and STATS is frequently used for calculating.

Compared with matched control, OXDC-CLEC oral medications can increase the survival rate of the AGT1KO mouse of EG attacks.Come 30 days conceptual phases are lived through from all mouse (11/11) of 80mgOXDC-CLEC treatment groups, without diseased sign, and are come from 4/11 mouse of control group has serious nephrocalcinosis, and develops urinary calculus.

Since OXDC-CLEC is intended for being administered orally, detrimental effect of the crosslinking crystals to stomach and intestine (GI) tissue is had rated Possibility.The alimentary canal of the AGT1 KO mouse of the EG attacks of visual inspection treatment, with stained with Hematoxylin-Eosin to including stomach Gastrointestinal tract different piece including (substance and sinus) and small intestine (jejunum and ileum) carries out histologic analysis.Evaluation confirmation, OXDC- CLEC oral medications 4 weeks are well-tolerateds, and will not cause the structure or metamorphosis of gastrointestinal tract.Class is observed for large intestine As result.

The summary studied with the dosage range of the EG of the oxalate decarboxylase-CLEC oral medications AGT1 KO mouse attacked.It is former OXDC-CLEC oral medications in hair property hyperoxaluria animal model are safe and efficient.It is controlled in short, OXDC-CLEC takes orally Treating makes urinary oxalate reduce 30-50% for 4 weeks.Using the experiment object of the evaluation of 3 dosage, significant and lasting reduction is recorded. Calcium oxalate deposits in 4 weekly assembly of highest therapeutic dose oral medication prevention kidney essence.In 2 lower dosage oral medications 4 weeks Survival rate can be improved, can prevent mortality of animals in highest research dosage.Finally, 4 weeks therapeutic schemes do not produce in the gastrointestinal tract Raw macroscopic view or microcosmic variation.

Embodiment 15. measures

Determination of protein concentration:By measuring absorbance in 280nm, the concentration of oxalate decarboxylase is measured.1.36 optical density (OD) absorbance is regarded as 1mg/ml.

OXDC determinations of activity:Use the improved SigmaAldrich schemes (enzymatic determination of oxalate decarboxylase EC4.1.1.2 Method), measure the work of soluble oxalate decarboxylase, oxalate decarboxylase crystal and crosslinked oxalate decarboxylase crystal (OXDC-CLEC) Property.This is a kind of indirect two steps determination of activity;In first reaction, OXDC is by substrate oxalate conversion formic acid salt and two Carbonoxide.In second reaction, formates hydrogen is transferred to NAD by formate dehydrogenase stochiometrically, is formed NADH.The successive concentrations of quantitative NADH in 340nm spectrophotometers.The unit (u) of enzymatic activity is defined as below:One unit Oxalate decarboxylase is per minute from 1.0 μm of ol oxalates formation formates and carbon dioxide at pH5 and 37 DEG C.

For OXDC crystal and OXDC-CLEC respectively in the concentration of 0.007-0.02 and 0.009-0.03 mg/mL, containing There are the 5mM kaliumphosphate buffer pH7.0 Plays determination samples of 1mMDTT (Sigma).By the absorbance in 280nm, survey Determine protein concentration.Before use in cold diH₂The formate dehydrogenase (FDH) of 40U/mL is prepared in O, is placed on ice.It is all its Its reagent is maintained at room temperature.In the following manner, reagent is added in into 2ml micro test tubes with stirring rod:Mix 300 μ l100mM phosphoric acid Potassium and 200 μ l potassium oxalate pH4.0, warm 5min at 37 DEG C, then 100 μ l are contained to the 5mM potassium phosphates of 1mM DTT in a water bath It adds in blank bottle, the diluted oxalate decarboxylases of 100 μ l is added in all other bottle.After 2 minutes, with 150mM phosphoric acid hydrogen two Potassium terminates reaction.In second reaction, 25 μ lNAD solution are added in into 100 μ lFDH solution, continue to incubate other 20min.So Afterwards in 16,100rpm centrifugation all samples 1min.Then reaction mixture is transferred to 1.5mL ultraviolet light test glass, used Shimadzu BioSpec (Shimadzu Scientific Instruments, Columbia, Maryland), measure The absorbance of 340nm, and record.

Enzyme is calculated as below than living:

In a specific experiment, crosslinked oxalate decarboxylase crystal (OXDC-CLEC) (is stayed overnight and 1% by overturning The rhomboidan of glutaraldehyde cross-linking;Referring to embodiment 9) it compares with OXDC crystal.OXDC-CLEC keeps corresponding lenticular The 50% of the activity of OXDC products.

Oxalates is measured by colourity method:Oxalates colorimetric kit for quantitative determining urine Oxalate is purchased from Trinity Biotech USA (St.Louis, MO) or Greiner Diagnostic AG (Dennliweg9, Switzerland).According to the specification of manufacturer, dilution and processing urine sample.It measures comprising 2 enzyme reactions:(a) oxalates oxygen Changing enzyme oxalates is oxidized to the hydrogen peroxide that carbon dioxide and hydrogen peroxide and (b) so formed is having peroxidase to deposit Lower with 3- methyl-2-benzothiazolinone hydrazones (MBTH) and 3- (dimethylamino) benzoic acid (DMAB) reaction, formation can be The indamines dyestuff of 590nm absorbances detection.The intensity of the color of generation and the concentration of sample Oxalate are directly proportional.From Standard curve calculates urinary oxalate value.

Kreatinin is measured by colourity method:Kreatinin colorimetric kit for quantitative determining kreatinin in urine is purchased from Quidel Corporation(San Diego,CA;METRA kreatinins assay kit) or Randox Laboratories (Antrim,United Kingdom).The measure is based on following principles, i.e. kreatinin is reacted with the picric acid in aqueous slkali, Form the product with the absorbance in 492nm.The amount of the compound of formation and Concentrations are directly proportional.With double distillations 15 times of the urine sample of rat for 24 hours that water dilution is collected from single metabolic cage.By the 20 diluted urine samples of μ l and 20 μ l picric acid/sodium hydroxide (1:1) mix.After incubation at room temperature 2 minutes, the absorbance in 492nm is measured.Urinary creatine acid anhydride value is calculated from standard curve.

Embodiment 16. is used to treat the OXDC treatments of the oxalates associated conditions of people

By taking orally crosslinked oxalate decarboxylase crystal, can treat needs to treat or prevent oxalates associated conditions for example The people of hyperoxaluria.It is applied in approximate dosage of the 10 μ g/kg to 25mg/kg, 1mg/kg to 25mg/kg or 5mg/kg to 100mg/kg With oxalate decarboxylase crystal, this is determined by treating clinician, and dependent on the seriousness of symptom and the progress of disease.It applies daily With the crosslinked crystal 1 of oxalate decarboxylase, 2,3,4 or 5 times or applied with lower frequency, such as once a week or 2 times.So Oral OXDC-CLEC cause urinary oxalate it is horizontal reduce at least 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70% or more.

The recombinant production of 17. oxalate decarboxylase of embodiment

In human embryo kidney (HEK293) cell:The DNA of OXDC will be encoded (for example, SEQIDNO:It 1 or 2) clones into suitable Expression vector in.After sequence confirms, by vector linearization, Lipofectamine is used in 6cm diameter disks^TM2000 transfections Reagent, with the HEK293 cells of the carrier conversion pre-vaccination of linearisation.Culture transfection reactant is stayed overnight in appropriate culture medium, so Afterwards transformant is selected in the culture medium for being with the addition of 0.5g/L neomycins.Growth up to 3 weeks in the culture medium containing neomycin Afterwards, the HEK293 cell clones of stable transfection are differentiated.Then clone is separated and bred, and is expressed for OXDC.

In Chinese hamster ovary (CHO) cell:The DNA clone of OXDC genes will be encoded into suitable expression vector. Then by Trypsin Induced, the CHOlec3.2.8.1 cells of culture are separated, is harvested by centrifugation.It then will be thin Born of the same parents are suspended in the brine buffer solution (EPBS) of electroporation phosphate-buffered to~1x10⁷The final concentration of/ml, is used by electroporation The carrier conversion of linearisation.After being incubated overnight, culture medium is changed to the culture medium for being with the addition of 0.5g/L neomycins.It is cultivated The continuous replacement of base, to screen the Chinese hamster ovary celI of stable transfection clone.Once the cell clone of stable transfection is established and breeds, it will Cell is expressed for OXDC.

In pichia pastoris:The DNA clone of OXDC genes will be encoded into suitable expression vector.Sequence confirms Afterwards, by vector linearization, be then transformed into pichia pastoris host cell (referring to, Whittaker et al., J.Biol.Inorg.Chem.7:136-145,2002).Transformant is selected with Zeocin, in the glycerol complex medium of buffering (BMGY) amplification in, and use methanol induction.Then OXDC can be separated from culture medium.

In saccharomyces cerevisiae:Can be by the OXDC gene clonings of synthesis into suitable expression vector, the latter is contained for example Gal1 promoters (pGal) and the terminator sequence of expression.After sequence confirms, expression vector is transformed by impression by electroporation State saccharomyces cerevisiae W303-1A.Transformant is screened, and is bred, is subsequently used for OXDC expression.

In insect cell:The DNA clone of OXDC can will be encoded into suitable expression vector, for example, baculoviral is System.After sequence confirms, carrier is transformed into competence DH10Bac Bacillus coli cells.It screens and confirms containing restructuring rod granule Bacillus coli cells.The bacmid dna of restructuring is separated, and uses reagent such as Cellfectin^TMReagent (Invitrogen, Carlsbad, California), for transfecting insect Sf 9 cells.Then recombinant baculovirus particle can be separated, is bred, And titrate, infection Sf9 cells are subsequently used for, carry out OXDC expression.

In Escherichia coli:The DNA clone of OXDC can will be encoded into suitable coli expression carrier.Sequence confirms Afterwards, carrier is transformed into competence e. coli bl21 or if necessary Escherichia coli Origami B (DE3), the latter allows Disulfide bond is formed in the recombinant protein expressed in the bacterial strain.By cultivating transformant on the nutrition tablet containing antibiotic, sieve Transformant is selected, and using the primer of OXDC gene specifics, is confirmed by bacterium colony PCR.Then train in liquid medium Transformant is supported, and is induced with isopropyl-β-D-thiogalactoside (IPTG), is expressed for OXDC.

18. sequence of embodiment

It will be in Candida boidinii（Candida boidinii）Needle mushroom sequence (the SEQIDNO of middle expression:1).2 Not I sequences are underlined;The ATA triplets of runic are interval codons;The sequence for indicating double underline is for Bo Yiding The α mating factor sequences of Candida optimization;OXDC coded sequence lowercase letters.

1 ataagaatGCGGCCGCATA

50

100

150

200

250 atgtttaataatt

300 ttcaaagattattaactgttattttattatctggttttactgctggtgtt

350 ccattagcttctactactactggtactggtactgctactggtacttctac

400 tgctgctgaaccatctgctactgttccatttgcttctactgatccaaatc

450 cagttttatggaatgaaacttctgatccagctttagttaaaccagaaaga

500 aatcaattaggtgctactattcaaggtccagataatttaccaattgattt

550 acaaaatccagatttattagctccaccaactactgatcatggttttgttg

600 gtaatgctaaatggccattttctttttctaaacaaagattacaaactggt

650 ggttgggctagacaacaaaatgaagttgttttaccattagctactaattt

700 agcttgtactaatatgagattagaagctggtgctattagagaattacatt

750 ggcataaaaatgctgaatgggcttatgttttaaaagggtctactcaaatt

800 tctgctgttgataatgaagggagaaattatatttctactgttggtccagg

850 tgatttatggtattttccaccaggtattccacattctttacaagctactg

900 ctgatgatccagaaggttctgaatttattttagtttttgattctggtgct

950 tttaatgatgatggtacttttttattaactgattggttatctcatgttcc

1000 aatggaagttattttaaaaaattttagagctaaaaatccagctgcttggt

1050 ctcatattccagctcaacaattatatatttttccatctgaaccaccagct

1100 gataatcaaccagatccagtttctccacaagggactgttccattaccata

1150 ttcttttaatttttcttctgttgaaccaactcaatattctggtgggactg

1200 ctaaaattgctgattctactacttttaatatttctgttgctattgctgtt

1250 gctgaagttactgttgaaccaggtgctttaagagaattacattggcatcc

1300 aactgaagatgaatggactttttttatttctggtaatgctagagttacta

1350 tttttgctgctcaatctgttgcttctacttttgattatcaaggtggtgat

1400 attgcttatgttccagcttctatgggtcattatgttgaaaatattggtaa

1450 tactactttaacttatttagaagtttttaatactgatagatttgctgatg

1500 tttctttatctcaatggttagctttaactccaccatctgttgttcaagct

1550 catttaaatttagatgatgaaactttagctgaattaaaacaatttgctac

1600 taaagctactgttgttggtccagttaattaaGCGGCCGCtaaactat1646

Bacillus subtilis sequence (SEQ ID NO:2):" G " that is underlined in position 705 refers to A → G bases Displacement.The base replacement will not change amino acid sequence.

1 ATGAAAAAACAAAATGACATTCCGCAGCCAATTAGAGGAGACAAAGGAG

50 CAACGGTAAAAATCCCGCGCAATATTGAAAGAGACCGGCAAAACCCTGAT

100 ATGCTCGTTCCGCCTGAAACCGATCATGGCACCGTCAGCAATATGAAGTT

150 TTCATTCTCTGATACTCATAACCGATTAGAAAAAGGCGGATATGCCCGGG

200 AAGTGACAGTACGTGAATTGCCGATTTCAGAAAACCTTGCATCCGTAAAT

250 ATGCGGCTGAAGCCAGGCGCGATTCGCGAGCTTCACTGGCATAAAGAAGC

300 TGAATGGGCTTATATGATTTACGGAAGTGCAAGAGTCACAATTGTAGATG

350 AAAAAGGGCGCAGCTTTATTGACGATGTAGGTGAAGGAGACCTTTGGTAC

400 TTCCCGTCAGGCCTGCCGCACTCCATCCAAGCGCTGGAGGAGGGAGCTGA

450 GTTCCTGCTCGTGTTTGACGATGGATCATTCTCTGAAAACAGCACGTTCC

500 AGCTGACAGATTGGCTGGCCCACACTCCAAAAGAAGTCATTGCTGCGAAC

550 TTCGGCGTGACAAAAGAAGAGATTTCCAATTTGCCTGGCAAAGAAAAATA

600 TATATTTGAAAACCAACTTCCTGGCAGTTTAAAAGATGATATTGTGGAAG

650 GGCCGAATGGCGAAGTGCCTTATCCATTTACTTACCGCCTTCTTGAACAA

700 GAGCCGATCGAATCTGAGGGAGGAAAAGTATACATTGCAGATTCGACAAA

750 CTTCAAAGTGTCTAAAACCATCGCATCAGCGCTCGTAACAGTAGAACCCG

800 GCGCCATGAGAGAACTGCACTGGCACCCGAATACCCACGAATGGCAATAC

850 TACATCTCCGGTAAAGCTAGAATGACCGTTTTTGCATCTGACGGCCATGC

900 CAGAACGTTTAATTACCAAGCCGGTGATGTCGGATATGTACCATTTGCAA

950 TGGGTCATTACGTTGAAAACATCGGGGATGAACCGCTTGTCTTTTTAGAA

1000 ATCTTCAAAGACGACCATTATGCTGATGTATCTTTAAACCAATGGCTTGC

1050 CATGCTTCCTGAAACATTTGTTCAAGCGCACCTTGACTTGGGCAAAGACT

1100 TTACTGATGTGCTTTCAAAAGAAAAGCACCCAGTAGTGAAAAAGAAATGC

1150 AGTAAATAA1158

Oxalate decarboxylase albumen (the Swiss-Prot as follows translated from bacillus subtilis sequence:O34714) (SEQIDNO:3)。

1 MKKQNDIPQPIRGDKGATVKIPRNIERDRQNPDMLVPPETDHGTVSNMK

50 FSFSDTHNRLEKGGYAREVTVRELPISENLASVNMRLKPGAIRELHWHKE

100 AEWAYMIYGSARVTIVDEKGRSFIDDVGEGDLWYFPSGLPHSIQALEEGA

150 EFLLVFDDGSFSENSTFQLTDWLAHTPKEVIAANFGVTKEEISNLPGKEK

200 YIFENQLPGSLKDDIVEGPNGEVPYPFTYRLLEQEPIESEGGKVYIADST

250 NFKVSKTIASALVTVEPGAMRELHWHPNTHEWQYYISGKARMTVFASDGH

300 ARTFNYQAGDVGYVPFAMGHYVENIGDEPLVFLEIFKDDHYADVSLNQWL

350 AMLPETFVQAHLDLGKDFTDVLSKEKHPVVKKKCSK 385

19. soluble OXDC of embodiment, stability of the lenticular OXDC and OXDC-CLEC in low pH3.0

By the soluble OXDC (embodiment 5) of 2.5mg/mL, the lenticular OXDC (embodiment 5) and 5.0mg/ of 5.0mg/mL The OXDC-CLEC (embodiment 10) of mL is placed in each 1mL sodium citrate buffer solutions pH3.0 in Eppendorf tube, in 37 °C of temperature Educate 5h.It is sampled when 0,2 and 5 are small, for measuring the stability of enzyme.Activity is measured as described in Example 15.Display 0,2 and 5 are small When after OXDC and OXDC-CLEC in the stability of pH3.0, the results are shown in Figure 7.Compared with initially, after pH3.0 incubates 5h, OXDC-CLEC retains about 100% activity, and soluble OXDC loses about 51% activity in preceding 2h, and about 40% work is only retained after 5h Property.Lenticular OXDC is more more stable than soluble OXDC, and about 68% activity is retained when 1 is small, and about 67% activity is retained when 2 is small.

20. soluble OXDC of embodiment, stabilization of lenticular OXDC and the OXDC-CLEC crystal in the presence of pepsin Property

By the soluble OXDC (embodiment 5) of 1.0mg/mL, 10.0mg/mL lenticular OXDC (embodiment 5) and The OXDC-CLEC (embodiment 10) of 1.0mg/mL is placed in each 1mL sodium citrate buffer solutions pH3.0 in Eppendorf tube, 37 °C, with OXDC:Pepsin is 50:1 ratio, with pepsin, (concentration of pepsin stoste is 1mg/mL, in 25mM In Tris-HCL buffer solutions, pH7.5) incubate 5h.It is sampled when 0,2 and 5 are small, for measuring the stability of enzyme.Such as embodiment 15 It is described to measure activity.Soluble OXDC, lenticular OXDC and OXDC-CLEC are with the presence of pepsin after when display 0,2 and 5 are small Under stability the results are shown in Figure 8.

As shown in figure 8, in low pH and in the presence of pepsin, disclose OXDC-CLEC preparations surpass it is soluble OXDC.Compared with initially, after 37 °C incubate 5h, OXDC-CLEC retains about 60% activity, and most of soluble OXDC and not Crosslinked lenticular OXDC, by Pepsin degradation, only retains about 20% activity after 2h after 5h.

21. soluble OXDC of embodiment, stability of the lenticular OXDC and OXDC-CLEC in the presence of the white enzyme of pancreas curdled milk

By the soluble OXDC (embodiment 5) of 1.0mg/mL, 10.0mg/mL lenticular OXDC (embodiment 5) and The OXDC-CLEC (embodiment 10) of 1.0mg/mL is placed in each 1mL25mM Tris-HCl buffer solutions in Eppendorf tube PH7.5, at 37 DEG C, with OXDC:Chymotrypsin is 50:1 ratio, with chymotrypsin (chymotrypsinogen liquid Concentration for 1mg/mL, in 25mM Tris-HCL buffer solutions, pH7.5, fresh preparation) incubate 5h.It is taken when 0,2 and 5 are small Sample, for measuring the stability of enzyme.Activity is measured as described in Example 15.Soluble OXDC, crystal after when display 0,2 and 5 are small The results are shown in Figure 9 for the stability of shape OXDC and OXDC-CLEC in the presence of trypsase.

Fig. 9's the result shows that, 37 DEG C incubate 5 it is small when after, OXDC-CLEC and uncrosslinked lenticular OXDC are stable , and it is resistant to the proteolysis cutting of chymotrypsin.Analysis shows, compared with the initial value before it is when 5 is small, OXDC-CLEC and uncrosslinked lenticular OXDC enzymes retain about 100% activity.Meanwhile when small exposed to chymotrypsin 2 Afterwards, soluble albumen loses about 50% activity, 5 it is small when after, all soluble OXDC are degraded, 0% activity of residue.

22. soluble OXDC of embodiment, the stabilization of lenticular OXDC and OXDC-CLEC in the simulated intestinal fluid containing pancreatin Property

By the soluble OXDC (embodiment 5) of 5.0mg/mL, 10.0mg/mL lenticular OXDC (embodiment 5) and Each 1mL simulated intestinal fluids that the OXDC-CLEC (embodiment 10) of 10.0mg/mL is placed in Eppendorf tube (are recommended to make according to USB Standby simulated intestinal fluid, is dissolved in 250ml water by 6.8gm potassium dihydrogen phosphates, is mutually mixed with 77ml0.2N sodium hydroxides and 500ml deionized waters It closes；Then, 10gm pancreatin is added in, pH is adjusted to pH6.8 with 0.2N hydrochloric acid or 0.2N sodium hydroxides；Add water to 1L), in 37 DEG C of temperature Educate 2h.It is sampled when 0,1 and 2 are small, for measuring the stability of enzyme.Activity is measured as described in Example 15.As a result such as Figure 10 institutes Show.

It is shown in Fig. 10 the result shows that, OXDC-CLEC be in the simulated intestinal fluid containing pancreatin it is stable, keep about 100% activity.On the contrary, most of soluble OXDC are interior by pancreatin (mixture of lipase, amylase and protease) when 1 is small Only residue about 26%-28% activity is distinguished in degradation after 1h and 2h is incubated.In the presence of pancreatin, uncrosslinked lenticular OXDC is more more stable than its soluble form, keeps about 76% and about 49% activity respectively after 1h and 2h is incubated.

In addition, OXDC-CLEC meeting protective enzymes are from protease（Such as pepsin and chymotrypsin）Cutting. Under the same terms, the stability of OXDC-CLEC can be the stability of soluble OXDC at least about 100%, 200%, 300%, 400% or more.Under the same conditions, the activity that OXDC-CLEC is maintained is than the activity up at least about 2,3 of soluble OXDC maintenances Or 4 times.Thus, compared with soluble OXDC, OXDC-CLEC is in acid pH of the scope from about 2.5 or 3 to about 7.5 or 8.5 It is active and is stable under pH and intestines harsh conditions containing different protease.

Many embodiments of the present invention have been described.It will nevertheless be understood that different modifications can be made, Without departing from the spirit and scope of the present invention.Therefore, other embodiments are also within the scope of the appended claims.

Claims (8)

1. the oxalate decarboxylase crystal of the bioactivity comprising spray drying for being administered orally is selected from sugar, sugar at least one Alcohol, tackifier, wetting agent, solubilizer, buffer salt, emulsifier, the capsule of excipient of antimicrobial and antioxidant are being made The application being ready for use in the drug of illness related with oxalates in treatment mammal, the illness choosing related with oxalates From hyperoxaluria and nephrocalcinosis, wherein the water content of the oxalate decarboxylase crystal for the bioactivity being spray-dried is less than 5 weights % is measured, condition is if oxalate decarboxylase crystal is crosslinked, then the crosslinking of oxalate decarboxylase crystal is in 0.05%w/v- It is carried out in the presence of the glutaraldehyde of 2.0%w/v.

2. application according to claim 1, wherein the hyperoxaluria is primary hyperoxaluria.

3. application according to claim 1, wherein the hyperoxaluria is intestines originality hyperoxaluria.

4. according to the application of any one of claim 1-3, wherein the oxalate decarboxylase crystal is uncrosslinked.

5. application according to claim 1, wherein the crystal is active in the gastrointestinal tract of mammal and is stable 's.

6. application according to claim 1, wherein the crystal is active in pH 2 to pH 8 and is stable.

7. application according to claim 1, wherein treating the illness related with oxalates includes oxalates reducing at least 15%.

8. application according to claim 1, wherein the glutaraldehyde cross-linking of the oxalate decarboxylase crystal 0.5%w/v.